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FINAL
ENGLISH ONLY
Recommendations to assure the quality, safety and efficacy of
live attenuated poliomyelitis vaccine (oral)
Replacement of: TRS 904, Annex 1 and Addendum TRS 910, Annex 1
© World Health Organization 2012
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Adopted by the 63rd
meeting of the WHO Expert Committee on Biological Standardization, 15 to 19
October 2012. A definitive version of this document, which will differ from this version in editorial but
not scientific details, will be published in the WHO Technical Report Series.
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Recommendations published by the WHO are intended to be scientific and advisory in nature.
Each of the following sections constitutes guidance for national regulatory authorities (NRAs)
and for manufacturers of biological products. If a NRA so desires, these Recommendations may
be adopted as definitive national requirements, or modifications may be justified and made by
the NRA. It is recommended that modifications to these Recommendations be made only on
condition that modifications ensure that the vaccine is at least as safe and efficacious as that
prepared in accordance with the recommendations set out below. The parts of each section
printed in small type are comments or examples for additional guidance intended for
manufacturers and NRAs, which may benefit from those details.
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Contents
Introduction ..................................................................................................................................... 4 Scope ........................................................................................................................................... 5
General considerations ................................................................................................................ 5 Part A. Manufacturing recommendations ................................................................................. 9
A.1 Definitions ........................................................................................................................ 9 A.2 General manufacturing recommendations ...................................................................... 12 A.3 Control of source material .............................................................................................. 12
A.4 Control of vaccine production ........................................................................................ 16 A.5 Filling and containers ..................................................................................................... 24
A.6 Control tests on the final lot ........................................................................................... 25 A.7 Records ........................................................................................................................... 27 A.8 Retained samples ............................................................................................................ 27 A.9 Labelling ......................................................................................................................... 27 A.10 Distribution and shipping ............................................................................................ 28
A.11 Stability, storage and expiry date ................................................................................ 28 Part B. Nonclinical evaluation of live attenuated poliomyelitis vaccines (oral) .................... 30
B.1 Characterization of a new virus sub-master seed from the WHO master seed .................. 30 B.2 Characterization of virus working seeds from an established master seed where passage
level between master and working seed is increased ................................................................ 30 B.3 Characterization following changes in the manufacturing process .................................... 30
Part C. Clinical evaluation of live attenuated poliomyelitis vaccines (oral) .......................... 32
C.1 General considerations ................................................................................................... 32
C.2 Safety and immunogenicity studies ................................................................................ 33 C.3 Post-marketing studies and surveillance ......................................................................... 35
Part D. Recommendations for national regulatory authorities ................................................ 36
D.1 General ............................................................................................................................ 36 D.2 Official release and certification ..................................................................................... 37
Part E. Recommendations for live attenuated poliomyelitis vaccine (oral) prepared in primary
cultures of monkey kidney ............................................................................................................ 38 E.4 Control of vaccine production ........................................................................................ 38
Authors and acknowledgements .................................................................................................... 44 References ..................................................................................................................................... 47
Appendix 1 ................................................................................................................................ 52 Appendix 2 ................................................................................................................................ 56
Appendix 3 ................................................................................................................................ 61 Appendix 4 ................................................................................................................................ 63 Appendix 5 ................................................................................................................................ 64 Appendix 6 ................................................................................................................................ 85 Appendix 7 ................................................................................................................................ 87
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Introduction
Requirements for oral poliomyelitis vaccine (OPV) were first formulated in 1962 (1) and revised
in 1966 (2) and 1972 (3) when an appendix describing the production of OPV in human diploid
cells was added. The requirements were further updated in 1982 (4) following an accumulation
of data, particularly on the performance and evaluation of the monkey neurovirulence test
(MNVT) and tests on the karyology of human diploid cells. The Requirements for poliomyelitis
vaccine (oral) were updated in full in 1989 (5) to take account of the general requirements for
the characterization of continuous cell lines for the preparation of biologicals which were
adopted in 1985 (6), and after a WHO Study Group concluded that, in principle, such cell lines
are acceptable as substrates for the production of biologicals (7). An addendum was
subsequently adopted (8) that introduced changes in tests for freedom from detectable DNA
sequences of Simian virus 40 (SV40), introduced the mutant analysis by PCR and restriction
enzyme cleavage (MAPREC) assay as an optional, additional in vitro test of poliovirus type 3,
increased levels of laboratory containment of wild polioviruses (9) and provided guidance on
additional antibody screening tests (for foamy viruses) for animals from closed primate colonies
used as a source for primary monkey kidney cells.
The requirements (now recommendations) were last revised in full in 1999 (10) when the use of
transgenic mice expressing the human poliovirus receptor (TgPVR21 mice) (11) as an alternative
to the MNVT for type 3 virus were included in the revision and the MAPREC test was
introduced as the in vitro test of preference for the evaluation of filtered bulk suspensions for
poliovirus type 3 (12). The rct40 test then became an optional, additional test. The studies with
poliovirus types 1 and 2 in TgPVR21 mice were completed by June 2000 and an addendum to
the Recommendations for the production and control of poliomyelitis vaccine (oral) was adopted
in 2000 (13) to include the neurovirulence test in TgPVR21 mice as an alternative to the MNVT
for all three poliovirus serotypes.
Since then there have been advances in scientific knowledge, novel laboratory techniques have
become available, and new vaccine formulations such as monovalent/bivalent OPV are being
used. In 2008, the WHO Expert Committee on Biological Standardization advised that the
recommendations for OPV should be revised. In addition, various tests are now applicable to all
three types of polioviruses and their significance needs to be better explained and rationalized.
Sections on nonclinical and clinical evaluation for new candidate OPVs are also required. To
facilitate this process, WHO convened a working group meeting to initiate the revision of the
recommendations for the production and control of OPV, as outlined in Nos. 904 and 910 of the
WHO Technical Report Series. Experts from academia, national regulatory authorities
(NRAs)/national control laboratories (NCLs) and industry involved in the research, manufacture,
authorization and testing/release of OPV from countries around the world met on 2022 July
2010 to identify and discuss the issues to be considered in the revision of the WHO Technical
Reports Nos. 904 and 910 (14).
Major issues addressed during this revision include:
an update on the origin of different strains for OPV production with inclusion of a
new Appendix 1;
an update of the section on international standards and reference preparations;
an update of the section on general manufacturing recommendations and control
tests;
an update on neurovirulence tests (MNVT and TgmNVT) and the MAPREC test
which is extended to all three types of seeds and bulks; and inclusion of a new
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Appendix 2 giving rationales for the choice of monkey or mouse neurovirulence
tests;
consideration of new vaccine formulations (mOPV and bOPV);
an update on terminology, and the introduction of the “virus sub-master seed lot”
which is applicable only to the master seed supplied by WHO;
inclusion of new sections on nonclinical and clinical evaluation of OPV;
an update on appendices;
an update on the standard operating procedures (SOPs) for TgmNVT and
MAPREC and new MNVT in light of current developments in techniques.
Additional changes have been made to bring the document into line with other WHO
recommendations published since the last revision.
Scope
The scope of the present recommendations encompasses live attenuated polio vaccines (oral)
derived from the original Sabin strains, some by simple passage and others by more complex
routes, including plaque purification. This document is intended to apply to all Sabin poliovirus
strains regardless of their history. It does not necessarily apply to other strains should they be
developed.
This document should be read in conjunction with the relevant WHO guidelines, such as those
on nonclinical (15) and clinical evaluation (16) of vaccines.
General considerations
Poliomyelitis is an acute communicable disease of humans caused by three distinct poliovirus
serotypes called types 1, 2 and 3 and distinguished by a neutralization test (17). Poliovirus is a
species C human enterovirus of the Picornaviridae family and is composed of a single-stranded,
positive-sense RNA genome and a protein capsid.
Where sanitation is poor, these viruses are believed to spread mainly by faecal-to-oral
transmission, whereas the oral-to-oral mode of transmission probably dominates in settings with
a higher standard of sanitation. However, in most settings, mixed patterns of transmission are
likely to occur. In the pre-vaccine era, roughly one out of 200 susceptible individuals infected by
polioviruses developed paralytic poliomyelitis (17).
Progress in polio control (and, since 1988, polio eradication) has been due mainly to widespread
use of vaccines. An inactivated poliovirus vaccine (IPV Salk vaccine) was licensed in 1955; live,
attenuated OPV (Sabin vaccine) was licensed as monovalent OPV in 1961 and as trivalent OPV
(tOPV) in 1963. The Sabin strains of poliovirus used in the production of OPV were shown to be
both immunogenic and highly attenuated when administered orally to susceptible children and
adults. Most countries that initially introduced vaccination with IPV later changed to OPV
because the latter provided many advantages, including ease of administration, suitability for
mass vaccination campaigns, superior induction of intestinal mucosal immunity, and lower
production costs. In 1974, OPV was recommended as part of the Expanded Programme on
Immunization (EPI), and OPV was again the vaccine of choice in 1988 when the World Health
Assembly resolved to eradicate polio globally by the year 2000. By 2010, three of the six WHO
regions had been certified as free of wild polio viruses, and wild type 2 has not been detected
worldwide since 1999 (17).
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In addition to tOPV, which is used in many countries for routine or supplementary vaccination,
monovalent OPVs against type 1 (mOPV1) and against type 3 (mOPV3) and bivalent OPV
against type 1 and type 3 (bOPV) (17), which are used by the Global Polio Eradication Initiative,
have been licensed for use in endemic countries or for outbreak control in situations where one
or two types can re-emerge. Monovalent OPV against type 2 has been licensed but is expected to
be used primarily for emergency response stockpiles. Recently, the Strategic Advisory Group of
Experts (SAGE) on Immunization was asked by WHO to consider the possible replacement of
tOPV with bOPV for routine immunization globally.
Following the introduction and widespread use of the first mOPV1 and mOPV3 in
supplementary immunization activities in 2005, the polio eradication programme has reported
substantial reductions in the respective poliovirus types. The last case in India was in January
2011 and, as polio is now considered to have been eradicated there, India has been removed from
the list of endemic countries. However, the co-circulation of wild poliovirus types 1 and 3 in the
three remaining polio-endemic countries requires huge quantities of bOPV to be used to
supplement tOPV given in routine and mass campaigns. A clinical trial to evaluate the
immunogenicity of different OPV formulations (mOPV1, mOPV3 and bOPV) compared to
tOPV in an Indian population was conducted by WHO. The seroconversion rates to poliovirus
type 1 and type 3 following immunization with bOPV were significantly higher than those
induced by tOPV and were not lower than those induced by immunization with either mOPV1 or
mOPV3 respectively (18).
Although OPV is a safe vaccine, adverse events may occur on rare occasions (17). Vaccine-
associated paralytic poliomyelitis (VAPP) is the most important of these rare adverse events.
Cases of VAPP are clinically indistinguishable from poliomyelitis caused by wild poliovirus, but
can be distinguished by laboratory analysis. The incidence of VAPP has been estimated at four
cases per million birth cohort per year in countries using OPV (19). Sabin viruses can spread in
populations where the coverage of OPV is low. In such situations, Sabin viruses can acquire the
neurovirulence and transmissibility characteristics of wild-strain poliovirus (WPV) and can
cause polio cases and outbreaks as circulating vaccine-derived poliovirus (cVDPV) (20).
Live vaccines prepared from the Sabin strains of poliomyelitis viruses of types 1, 2 and 3 were
introduced for large-scale immunization in 1957. In 1972, Sabin proposed that WHO should be
the custodian of his poliovirus seed strains. The Director-General of WHO agreed to assume
responsibility for ensuring the proper use of the strains and established a scientific committee,
the Consultative Group on Poliomyelitis Vaccines, to advise WHO on all matters pertaining to
their use. Detailed information on the work of the Consultative Group and the preparation of the
strains prepared by Behringwerke has been published by Cockburn (21). NRAs should decide on
the use of virus strains and on the detailed procedures applicable to the preparation of virus seed
lots for the production of OPV in their own countries.
The original poliovirus seeds produced by Sabin (SO) (22) were sent to Merck which generated
seeds from them that were designated as SOM (Sabin Original Merck). Aliquots of SOM were
supplied to a number of other manufacturers to enable them to develop their own seeds. Some
seed lots were contaminated with SV40 which was present in the primary Rhesus kidney cells,
the preferred cell culture system at that time for virus propagation. OPV manufacturers followed
various strategies to reduce the contamination, including passage in the presence of specific
antibody or treatment with toluidine blue, or thermal inactivation of SV40 in the presence of 1M
MgCl2 that stabilizes poliovirus. In 1974 Behringwerke AG of Marburg/Lahn, Germany,
generously agreed to produce SO+1 seeds for WHO free of charge. The Behringwerke type 1
and type 2 seeds have been particularly widely used from the 1970s to date.
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In the 1950s, it was established that, particularly for the type 3 strain, increase in the passage
number correlated with an increase in the reactivity in the MNVT. This finding led to the
establishment of rigorous limits on the passage level for vaccine production for all types.
The type 3 vaccine was found to be less stable on passage than either type 1 or type 2, which was
manifested in a higher number of type 3 vaccine lots failing the monkey neurovirulence test. In
order to develop a more stable strain, a new seed was prepared by Pfizer by transfecting
susceptible cells with viral RNA extracted from poliovirus at the SO+2 level. One plaque, which
was designated 457-III, was identified with particularly favourable properties (23). Theoretically,
vaccine derived from this stock was at passage SO+7. However, the purpose of tracking passage
history of seed viruses is to reduce the accumulation of mutations that takes place in the course
of their serial propagation. Since plaque purification represents the cloning of a single infectious
particle, it eliminates the heterogeneity of viral population and the passage level is effectively
reset to zero. Thus the cloned stock 457-III was renamed RSO (for RNA-derived Sabin Original).
Two additional passages were used to prepare virus master (RSO1) and working seeds (RSO2),
and vaccines produced from this virus are at RSO3 level. Retrospectively, the sequence of RSO
has been shown to be the same as the consensus of SO (24), but it was more homogeneous and
contained lower quantities of mutant viruses.
The RSO seed was not used for the production of type 3 vaccine until the 1980s when it became
clear that the stocks of material passaged from the SOM and other SO+1 seeds were inadequate.
Since then, however, it has been widely used by European and American manufacturers as it is
of lower virulence in laboratory tests than the SO+1 type 3 seed. The RSO seeds were bought
from Pfizer by Sanofi Pasteur (formerly Merieux, Pasteur Merieux Connaught and subsequently
Aventis Pasteur) which has recently donated them to WHO.
The virus seeds available from WHO (designated “the WHO master seeds”) are therefore types 1,
2 and 3 at SO+1 level produced by Behringwerke from SO seeds and the type 3 RSO seed
donated by Sanofi Pasteur. The seeds are kept at the National Institute for Biological Standards
and Control in the United Kingdom, and include a proportion of the stocks of the SO+1 seeds
formerly held at Istituto Superiore di Sanità in Italy which kindly transferred them (21, 23).
In addition to the RSO type 3 seed, a number of manufacturers in China, Japan and Russia have
used their own purified seed stocks of Sabin 3 strain that were derived by plaque purification
(cloning). Sequencing of these seed viruses demonstrated that, while they contained low content
of neurovirulent mutants, there were differences between these strains and the consensus
sequence of Sabin Original virus (24). However, there are no reports of any differences in
clinical safety between OPV produced from Pfizer stocks and the alternative seeds of Sabin 3
virus. An overview of virus seeds used in OPV production is given in Appendix 1.
The MNVT, as described in the 1989 requirements (5), has been used as a quality control test
and is based on the level and the distribution of virus-specific lesions within the central nervous
system produced by vaccine virus as compared to an appropriate reference preparation (25).
Because nonhuman primates are used, efforts to complement and eventually replace the test are
of considerable importance. WHO has encouraged and supported research on various aspects of
poliovirus biology, including the development of alternative animal models, as part of the WHO
initiative to promote the development of new norms and standards for vaccines. Two groups of
scientists developed transgenic (TgPVR) mice by introducing into the mouse genome the human
gene encoding the cellular receptor for poliovirus (26, 27). This receptor, known as CD155,
makes TgPVR mice susceptible to poliovirus infection with clinical signs of flaccid paralysis and
with histological lesions in the central nervous system similar to those observed in monkeys.
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In 1992, WHO initiated a project to evaluate the suitability of transgenic mice for testing the
neurovirulence of OPV with the aim of replacing monkeys with mice. The advantages of a
neurovirulence test in transgenic mice are:
a reduction in the number of primates used in quality control of OPV;
the use of animals of highly-defined genetic and microbiological quality
standards;
a reduction in hazards to laboratory personnel through a reduced need to
handle primates;
in some countries, a reduction in the cost of quality control tests for OPV.
Studies were carried out initially on type 3 monovalent polio vaccines using the TgPVR21
mouse line, generously provided free of charge for the study by the Central Institute for
Experimental Animals in Kawasaki, Japan. Researchers at the Japan Poliomyelitis Research
Institute and at the Center for Biologics Evaluation and Research (CBER) in Rockville, MD,
USA developed an intraspinal inoculation method suitable for tests of vaccine lots. This was
evaluated in an international collaborative study on the establishment of a standardized mouse
neurovirulence test (TgmNVT) for OPV (28). Several laboratories participated in the
collaborative study and results were assessed by WHO at meetings held in 1995, 1997 and 1999
in Geneva, Switzerland, in 1997 in Ottawa, Canada, and in 1998 in Rockville, MD, USA. As a
result of these studies, the revised Recommendations for the production and control of
poliomyelitis vaccine (oral) (10) introduced the murine model as an alternative to the MNVT for
type 3 poliovirus and further studies demonstrated that this test was also suitable as an alternative
to the MNVT for poliovirus types 1 and 2 (13). Laboratories must comply with specifications for
containment of the transgenic animals (29). The MNVT and TgmNVT can provide evidence of
consistency of production.
The molecular mechanisms and genetic determinants of attenuation and reversion to virulence of
all three types of Sabin polioviruses used for the manufacture of OPV have been studied in
several laboratories. Evidence strongly suggests that mutations in the 5’ noncoding region of the
poliovirus genome, especially for the Sabin type 3 strain, are critical in determination of the
attenuated phenotype (30). A molecular biological test, MAPREC assay, was developed by
researchers at CBER to quantify reversion at the molecular level (31). Studies showed that all
batches of type 3 OPV contained measurable amounts of revertants with C instead of U at
nucleotide 472. Batches that failed the MNVT contained significantly higher quantities of 472-C
than batches that passed the test. Studies with coded samples at CBER identified 100% of lots
that failed the MNVT (32).
In 1991, WHO initiated a series of international collaborative studies to evaluate the MAPREC
assay for all three types of poliovirus and to validate appropriate reference materials. Several
laboratories participated in the collaborative studies and results were assessed by WHO at
meetings held in 1995 and 1997 in Geneva, Switzerland. It was concluded that the MAPREC
assay was a sensitive, robust and standardized molecular biological assay suitable for use by
manufacturers and NRAs for monitoring the consistency of production of type 3 OPV. The
revised Recommendations for the production and control of poliomyelitis vaccine (oral) (10)
introduced, for type 3 poliovirus, the use of MAPREC as the in vitro test of preference in place
of the rct40 test. Reference materials for MAPREC for comparable positions in type 1 and type 2
have now been established. While the results do not correlate with neurovirulence in the range
studied, they provide a measure of production consistency. The quantity of other mutants (such
as 2493-U in Sabin 3 virus) can also be used to identify types of seed virus and to monitor
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consistency of manufacture. After appropriate validation, quantitative profiles of other mutations
in stocks of OPV could be used for this purpose.
The manufacturer of the final lot must be responsible for ensuring conformity with all the
recommendations applicable to the final vaccine (Part A, sections A.5A.11) even where
manufacturing involves only the filling of final containers with vaccine obtained in bulk form
from another manufacturing establishment. The manufacturer of the final lot must also be
responsible for any production and control tests performed by an external contract laboratory, if
applicable, with the approval of the NRA.
OPV has been in worldwide use since the 1960s and, although the vaccines produced from
human diploid cells or continuous cell lines have been used to a lesser extent than those
produced in cultures of primary monkey kidney cells, experience has indicated that all three cell
substrates produce safe and effective vaccines.
In 1986, a WHO study group (7) stated that the risks for residual cellular DNA (rcDNA) for
vaccines produced in continuous cell lines should be considered negligible for preparations given
orally. This conclusion was based on the finding that polyoma virus DNA was not infectious
when administered orally (33). For such products, the principal requirement is the elimination of
potentially contaminating viruses. Recently, additional data on the uptake of DNA via the oral
route have been published (34). These studies demonstrated that the efficiency of uptake of DNA
introduced orally was significantly lower than that of DNA introduced intramuscularly.
Nevertheless, the specifics of the manufacturing process and the formulation of a given product
should be considered by the NRA (35) and, where possible, data should be accumulated on the
levels of rcDNA in OPV produced in Vero cells.
At the time of preparation of this document, there was increasing interest in developing, through
molecular manipulation, alternative strains for use in OPV production. The poliovirus-specific
quality evaluation of such strains – e.g. neurovirulence testing, MAPREC etc – as described in
these recommendations and associated SOPs, may not be appropriate. The tests on such vaccines,
which are likely to include extensive preclinical and clinical studies to demonstrate attenuation,
genetic stability, safety and transmissibility of the proposed strains, will have to be considered
separately on a case-by-case basis and may differ fundamentally from those described in this
document.
Part A. Manufacturing recommendations
A.1 Definitions
A.1.1 International name and proper name
The international name should be live attenuated poliomyelitis vaccine (oral) with additions to
indicate the virus serotype(s) of the vaccine. The proper name should be equivalent to the
international name in the language of the country of origin.
The use of the international name should be limited to vaccines that satisfy the specifications
formulated below.
A.1.2 Descriptive definition Live attenuated poliomyelitis vaccine (oral) is a preparation of live attenuated poliovirus types 1,
2 or 3 grown in in vitro cultures of suitable cells, containing any one type or any combination of
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the three types of the Sabin strains, prepared in a form suitable for oral administration and
satisfying all the recommendations formulated in this document.
A.1.3 International reference materials
A trivalent virus mixture is available as an International Reference Reagent for Live Attenuated
Poliovirus (Sabin) Types 1, 2 and 3 for determination of virus titre.
Three monotypic virus suspensions of types 1, 2 and 3 have been established as WHO reference
reagents for use in reference laboratories to measure the sensitivity of cell cultures for poliovirus
infection.
International Standards for MAPREC analysis of poliovirus types 1, 2 and 3 (Sabin) and
International Reference Reagents for control of MAPREC assays of poliovirus type 1, 2 and 3
(Sabin) are available.
International Standards for anti-poliovirus types 1, 2 and 3 antibodies (human) are available for
standardization of neutralizing antibody tests for poliovirus.
The reference materials listed above are available from the
National Institute for Biological Standards and Control,
Potters Bar, United Kingdom.
Reference preparations at the SO+2 passage level, designated WHO/I for type 1 virus, WHO/II
for type 2 virus and WHO/III for type 3 virus, are available upon request from WHO.1 These
reference preparations are for use in in vivo neurovirulence tests with homotypic vaccines. The
relevant reference materials should be included in each test of vaccine (see section A.4.4.7.2).
A.1.4 Terminology The definitions given below apply to the terms as used in these recommendations. They may
have different meanings in other contexts.
Adventitious agents: Contaminating microorganisms of the cell substrate or source materials
used in their cultures, that may include bacteria, fungi, mycoplasmas, and endogenous and
exogenous viruses that have been unintentionally introduced.
Cell-culture infective dose 50% (CCID50): The quantity of a virus suspension that will infect
50% of cell cultures.
Cell seed: A quantity of vials containing well-characterized cells derived from a single tissue or
cell of human or animal origin stored frozen in liquid nitrogen in aliquots of uniform
composition, one or more of which would be used for the production of a master cell bank.
Comparator vaccine: An approved vaccine with established efficacy or with traceability to a
vaccine with established efficacy that is tested in parallel with an experimental vaccine and
serves as an active control in nonclinical or clinical testing.
Final bulk: The finished vaccine from which the final containers are filled. The final bulk may
be prepared from one or more monovalent bulks and may contain more than one virus type.
1 Contact the Coordinator, Quality, Safety and Standards, World Health Organization, 1211, Geneva 27, Switzerland
(http://www.who.int/biologicals/vaccines/en/).
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Final lot: A collection of sealed final containers of finished vaccine that is homogeneous with
respect to the risk of contamination during the filling process. All of the final containers must
therefore have been filled from a single vessel of final bulk in one working session.
Master cell bank (MCB): A quantity of fully characterized cells of human or animal origin
frozen at 70 °C or below in aliquots of uniform composition, derived from the cell seed. The
MCB is itself an aliquot of a single pool of cells, dispensed into multiple containers and stored
under defined conditions. The MCB is used to derive all working cell banks. The testing
performed on a replacement MCB – derived from the same cell clone, or from an existing master
or working cell bank (WCB) – is the same as that for the initial master cell bank unless a
justified exception is made.
Monovalent bulk: A pool of a number of single harvests of the same virus type.
Production cell culture: A cell culture derived from one or more ampoules of the WCB or
primary tissue used for the production of vaccines.
RSO: RNA-derived Sabin Original type 3 virus (23). All subsequent passages are designated by
an additional number – e.g. RSO1 (master seed) is one passage on from RSO. The working seed
passage level is therefore RSO2 and the vaccine RSO3.
Single harvest: A quantity of virus suspension of one virus type harvested from cell cultures
derived from the same WCB and prepared from a single production run.
SO: Sabin Original virus as described in Sabin and Boulger 1983 (22). All subsequent passages
are designated by an additional number – e.g. SO+1 is one passage on from Sabin Original.
Virus master seed lot: A quantity of virus suspension that has been processed at the same time to
assure a uniform composition and has been characterized to the extent necessary to support
development of the virus working seed lot. The characterized virus master seed lot is used for the
preparation of virus working seed lots or a virus sub-master seed (if applicable).
Virus sub-master seed lot (only applicable for master seed supplied by WHO): A quantity of
virus suspension produced by a single passage from the virus master seed supplied by WHO and
made at a multiplicity of infection that ensures the development of cytopathic effect within an
appropriate timeframe, and that has been processed at the same time to assure a uniform
composition. The virus sub-master seed lot should be characterized to the extent necessary to
support the development of the virus working seed lot. The characterized virus sub-master seed
lot is used for the preparation of virus working seed lots (see section A.3.2.2 and Part B).
Virus working seed lot: A quantity of virus of uniform composition, fully characterized, derived
by only one passage made at the multiplicity of infection, ensuring that cytopathic effect
develops within an appropriate timeframe (e.g. three days) from a virus master seed lot or sub-
master seed lot by a method approved by the NRA.
Working cell bank (WCB): A quantity of cells of uniform composition derived from one or
more ampoules of the MCB at a finite passage level, stored frozen at –70 °C or below in aliquots,
one or more of which would be used for vaccine production. All containers are treated
identically and once removed from storage are not returned to the stock.
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A.2 General manufacturing recommendations
The general manufacturing recommendations contained in Good manufacturing practices for
pharmaceutical products: main principles (36) and Good manufacturing practices for biological
products (37) should apply to establishments manufacturing OPV, with the addition of the
following:
The production of OPV should be conducted by staff who should be healthy persons
and who should be examined medically at regular intervals. Steps should be taken to
ensure that all such persons in the production areas are immune to poliomyelitis.
Personnel working in monkey quarters should also be examined for tuberculosis as
outlined in Part A, section 2 of Recommendations to assure the quality, safety and
efficacy of freeze-dried BCG vaccine (38).
The establishment should be in compliance with the current global recommendations
for poliovirus containment.
A.3 Control of source material
General production precautions, as formulated in Good manufacturing practices for biological
products (37) should apply to the manufacture of OPV, with the addition that, during production,
only one type of cell should be introduced or handled in the production area at any one time.
Vaccines may be produced in cell lines such as MRC-5 and Vero cells (see section A.3.1) or in
primary monkey kidney cells (PMKC) (see Part E).
A.3.1 Cell lines
A.3.1.1 Master cell bank (MCB) and working cell bank (WCB)
The use of a cell line for the manufacture of OPVs should be based on the cell bank system. The
cell seed and cell banks should conform with the Recommendations for the evaluation of animal
cell cultures as substrates for the manufacture of biological medicinal products and for the
characterization of cell banks (35). The cell bank should be approved by the NRA. The
maximum number of passages (or population doublings) allowed between the cell seed, the
MCB, the WCB and the production passage level should be established by the manufacturer and
approved by the NRA. Additional tests may include, but are not limited to: propagation of the
MCB or WCB cells to or beyond the maximum in vitro age for production, and examination for
the presence of retrovirus and tumorigenicity in an animal test system (35).
It is important to show that the cell banks (cell seed, MCB and WCB) are free of adventitious
agents relevant to the species used in their derivation. Cell banks should be assessed for the
absence of adventitious agents that may have been present during production.
The WHO Vero reference cell bank 10-87 is considered
suitable for use as a cell seed for generating an MCB (39) and
is available to manufacturers on application to the
Coordinator, Quality, Safety and Standards, World Health
Organization, Geneva, Switzerland.
A.3.1.2 Identity test
Identity tests on the master (MCB) and working cell banks (WCB) are performed in accordance
with WHO’s Recommendations for the evaluation of animal cell cultures as substrates for the
Page 13
manufacture of biological medicinal products and for the characterization of cell banks (35) and
should be approved by the NRA.
The WCB should be identified by means of, inter alia, biochemical tests (e.g. isoenzyme
analysis), immunological tests, cytogenetic marker tests and DNA fingerprinting or sequencing.
The tests should be approved by the NRA.
A.3.1.3 Cell culture medium
Serum used for the propagation of cells should be tested to demonstrate freedom from bacterial,
fungal and mycoplasma contamination by appropriate tests as specified in Part A, sections 5.2
(40) and 5.3 (41) of the General requirements for the sterility of biological substances, and
freedom from infectious viruses. Suitable tests for detecting viruses in bovine serum are given in
Appendix 1 of WHO’s Recommendations for the evaluation of animal cell cultures as substrates
for the manufacture of biological medicinal products and for the characterization of cell banks
(35).
Validated molecular tests for bovine viruses may replace the cell culture tests of bovine sera if
approved by the NRA. As an additional monitor of quality, sera may be examined for freedom
from phage and endotoxin. Gamma irradiation may be used to inactivate potential contaminant
viruses, recognizing that some viruses are relatively resistant to gamma irradiation.
The source(s) of animal components used in the culture medium should be approved by the NRA.
These components should comply with the current WHO guidelines on transmissible spongiform
encephalopathies in relation to biological and pharmaceutical products (42).
Human serum should not be used. If human serum albumin is used at any stage of product
manufacture, the NRA should be consulted regarding the requirements, as these may differ from
country to country. As a minimum, the serum should meet the Requirements for the collection,
processing and quality control of blood, blood components and plasma derivatives (43). In
addition, human albumin and materials of animal origin should comply with current WHO
guidelines on transmissible spongiform encephalopathies in relation to biological and
pharmaceutical products (42).
Penicillin and other beta-lactams should not be used at any stage of manufacture because of their
nature as highly sensitizing substances.
Other antibiotics may be used at any stage in the manufacture
provided that the quantity present in the final lot is acceptable
to the NRA.
Nontoxic pH indicators may be added, such as phenol red in
a concentration of 0.002%.
Only substances that have been approved by the NRA may be
added.
Bovine or porcine trypsin used for preparing cell cultures should be tested and found free of
cultivable bacteria, fungi, mycoplasmas and infectious viruses, as appropriate. The methods used
to ensure this should be approved by the NRA.
In some countries, irradiation is used to inactivate potential
contaminant viruses. If irradiation is used, it is important to
Page 14
ensure that a reproducible dose is delivered to all batches and
to the component units of each batch. The irradiation dose
must be low enough for the biological properties of the
reagents to be retained but also high enough to reduce
virological risk. Therefore, irradiation cannot be considered a
sterilizing process (35).
Recombinant trypsin is available and should be considered;
however, it should not be assumed to be free from risk of
contamination and should be subject to the usual
considerations for any reagent of biological origin (35).
The source(s) of trypsin of bovine origin, if used, should be approved by the NRA and should
comply with the current WHO guidelines on transmissible spongiform encephalopathies in
relation to biological and pharmaceutical products (42).
A.3.2 Virus seeds
A.3.2.1 Virus strains
Strains of poliovirus used in the production of OPV should be identified by historical records,
which should include information on their origin. Producers of OPV can obtain virus master
seeds from WHO. Manufacturers receiving this virus may prepare a sub-master seed by a single
passage and then prepare their working seed. However, only virus strains that are approved by
the NRA should be used (see General considerations).
A.3.2.2 Virus seed lot system
Vaccine production should be based on the seed lot system. Virus seed lots should not be
purified. The virus master seed lot and virus working seed lot used for the production of vaccine
batches should be prepared by a single passage from the virus strain and the virus master seed lot
respectively, by a method and at a passage level from the original seed virus approved by the
NRA. A virus sub-master seed lot may be prepared by a single passage from the WHO master
seed, and the characterized virus sub-master seed lot (see Part B) may be used for the preparation
of virus working seed lots by a single passage.
Virus master, sub-master and working seed lots should be stored in dedicated temperature-
monitored freezers at a temperature that ensures stability on storage e.g. ≤ 60 ºC. Guidance on
additional characterization of master and sub-master seeds is provided in Part B.
A.3.2.3 Tests on virus master, sub-master and working seed lots
The virus master seed is provided by WHO as well characterized seed material. The virus sub-
master and working seed lot used for the production of vaccine batches should be shown to be
free from detectable extraneous viruses and from detectable SV40 DNA, as determined by a
validated nucleic acid amplification test, and should be in conformity with the recommendations
set out in Part A, sections A.4.3 (Single harvests) and A.4.4.1, A.4.4.2, A.4.4.3 and A.4.4.4
(Monovalent bulks). The control cell cultures should conform to section A.4.1 (Control of cell
cultures).
DNA of SV40 is widely used as molecular biological reagent,
and contamination of polymerase chain reaction (PCR)
assays is potentially a major problem. One approach is to
identify separate genomic regions of SV40 for amplification,
Page 15
and to use one region for screening purposes and the other for
the confirmation of repeatedly positive samples. It is useful if
the second genomic region used for confirmation varies
between isolates from different sources, as it is then possible
to show that it has a unique sequence and that positive results
are not due to contamination with laboratory strains of SV40.
The sensitivity of the PCR assays for the genomic regions
used should be established.
A.3.2.4 Tests to monitor virus molecular characteristics
A.3.2.4.1 Tests in vitro
Seed viruses should be tested in MAPREC or temperature sensitivity assays (rct40) (see section
A.4.4.7.1). At least three consecutive monovalent bulks prepared from the seed virus should
meet the criteria for acceptability given in section A.4.4.7.1, with the agreement of the NRA.
Historically, four consecutive monovalent bulks prepared
from the seed virus have been tested for monitoring the virus
molecular characteristics and production consistency.
A.3.2.4.2 Neurovirulence tests
New virus working seeds should be evaluated for neurovirulence. Summaries of the MNVT and
TgmNVT, including pass/fail criteria, are given in Appendix 2 along with considerations on the
choice of assay. The test should be approved by the NRA for the specific product, and transgenic
mice, nonhuman primates, or both may be used.
The test for neurovirulence in nonhuman primates should be carried out as summarized in
Appendix 2 and following the SOP Neurovirulence test of types 1, 2 or 3 live attenuated
poliomyelitis vaccines (oral) in monkeys, available from WHO.1
The use of the TgmNVT should be approved by the NRA and it should be carried out as
summarized in Appendix 2 and described in detail in the SOP Neurovirulence test of type 1, 2 or
3 live attenuated poliomyelitis vaccines (oral) in transgenic mice susceptible to poliovirus,
available from WHO (see above).
Under normal circumstances, a new virus working seed will be prepared using the same
production protocol and from the same virus master seed as the currently approved virus
working seed. If the TgmNVT has been approved by the NRA for the release of vaccine batches,
and if the virus working seed is generated by the same production process, the new seed can be
qualified by use of the transgenic mouse test and supporting in vitro data alone.
In case there are any major changes in the production process for a new virus master seed, full
characterization in tests in nonhuman primates and transgenic mice will be required (See Part B).
The neurovirulence of the virus working seeds and at least three consecutive monovalent bulks
prepared from it should meet the criteria for acceptability given in section A.4.4.7.2 and the
appropriate SOP before the virus working seed can be considered suitable for use for the
production of OPV, with agreement of the NRA.
1 Contact the Coordinator, Quality, Safety and Standards, World Health Organization, 1211, Geneva 27, Switzerland
(http://www.who.int/biologicals/vaccines/en/).
Page 16
Historically, four consecutive monovalent bulks prepared
from the seed virus have been tested in monkeys for
monitoring the production consistency.
A.3.2.5 Genotype characterization
Advances have been made in the development and
application of molecular methods such as deep sequencing.
For any new virus working seed, it may be useful for
information purposes to analyse the new virus working seed
and at least three consecutive monovalent bulks for
nucleotide sequence changes from the seed virus (deep
genome sequence). If such tests are performed for regulatory
purposes, they should be scientifically validated and
approved by the NRA.
A.4 Control of vaccine production
Part E contains additional or alternative recommendations for OPV prepared in cultures of
primary monkey kidney cells and concerns the testing of the cell substrate used for the
production of the vaccine.
A.4.1 Control cell cultures
When human diploid or continuous cell lines are used to prepare cultures for the production of
vaccine, a fraction equivalent to at least 5% of the total or 500 ml of cell suspension, or 100
million cells, at the concentration and cell passage level employed for seeding vaccine
production cultures, should be used to prepare control cultures. (See Appendix 3 for an example
of a flowsheet of tests in cell cultures).
If fermenter technology is used, the NRA should determine the size and treatment of the cell
sample to be examined.
A.4.1.1 Tests of control cell cultures
The treatment of the cells set aside as control material should be similar to that of the production
cell cultures, but they should remain uninoculated for use as control cultures for the detection of
adventitious agents.
These control cell cultures should be incubated under conditions as similar as possible to the
inoculated cultures for at least two weeks, and should be tested for the presence of adventitious
agents as described below. For the test to be valid, not more than 20% of the control cell cultures
should have been discarded for nonspecific, accidental reasons.
At the end of the observation period, the control cell cultures should be examined for
degeneration caused by an extraneous agent. If this examination, or any of the tests specified in
this section, shows evidence of the presence in a control culture of any adventitious agent, the
poliovirus grown in the corresponding inoculated cultures should not be used for vaccine
production.
A.4.1.2 Tests for haemadsorbing viruses
At the end of the observation period, 25% of the control cells should be tested for the presence of
haemadsorbing viruses using guinea pig red blood cells. If these cells have been stored, the
Page 17
duration of storage should not have exceeded seven days and the storage temperature should
have been in the range 28 °C. In tests for haemadsorbing viruses, calcium and magnesium ions
should be absent from the medium.
Some NRAs require, as an additional test for haemadsorbing
viruses, that other types of red cells, including cells from
humans (blood group IV O), monkeys and chickens (or other
avian species), should be used in addition to guinea pig cells.
A reading should be taken after 30 minutes’ incubation at 28 °C and again after a further
incubation for 30 minutes at 2025 °C.
If a test with monkey red cells is performed, readings should
also be taken after a final incubation for 30 minutes at
3437 °C.
A.4.1.3 Tests for other adventitious agents in cell fluids
At the end of the observation period, a sample of the pooled fluid from each group of control
cultures should be tested for adventitious agents. For this purpose, 10 ml of each pool should be
tested in the same cells, but not the same batch of cells, as those used for the production of
vaccine.
A second indicator cell line should be used to test an additional 10 ml sample of each pool.
When a human diploid cell line is used for production, a simian kidney cell line should be used
as the second indicator cell line. When a simian kidney cell line is used for production, a human
diploid cell line should be used as the second indicator cell line (35).
The pooled fluid should be inoculated into bottles of these cell cultures in such a way that the
dilution of the pooled fluid in the nutrient medium does not exceed 1 part in 4. The area of the
cells should be at least 3 cm2 per ml of pooled fluid. At least one bottle of each kind of cell
culture should remain uninoculated and should serve as a control.
The inoculated cultures should be incubated at a temperature of 3537 °C and should be
observed for a period of at least 14 days.
Some NRAs require that, at the end of this observation period,
a subculture is made in the same culture system and observed
for at least an additional 14 days. Furthermore, some NRAs
require that these cells should be tested for the presence of
haemadsorbing viruses.
For the tests to be valid, not more than 20% of the culture vessels should have been discarded for
nonspecific, accidental reasons by the end of the test period.
If any cytopathic changes due to adventitious agents occur in any of the cultures, the virus
harvests produced from the batch of cells from which the control cells were taken should be
discarded.
Some selected viruses may be screened by using specific validated assays which are approved by
the NRA, such as molecular techniques (e.g. nucleic acid amplification) (35).
Page 18
If these tests are not performed immediately, the samples should be kept at a temperature of
60 °C or below.
A.4.1.4 Identity test
At the production level, the cells should be identified by means of tests approved by the NRA.
Suitable methods are, but are not limited to, biochemical tests (e.g. isoenzyme analyses),
immunological tests, cytogenetic tests (e.g. for chromosomal markers) and tests for genetic
markers (e.g. DNA fingerprinting or sequencing).
A.4.2 Cell cultures for vaccine production
A.4.2.1 Observation of cultures for adventitious agents
On the day of inoculation with the virus working seed lot, each cell culture or a sample from
each culture vessel should be examined visually for degeneration caused by infective agents. If
such examination shows evidence of the presence in a cell culture of any adventitious agent, the
culture should not be used for vaccine production (see section A.4.1.3).
If animal serum is used for cell cultures before the inoculation of virus, the medium should be
removed and replaced with serum-free maintenance medium after the cells have been washed
with serum-free medium, if appropriate.
A.4.3 Control of single harvests
A.4.3.1 Single harvest
After inoculation of the production cells with the virus working seed lot, inoculated and control
cell cultures should be held at a fixed temperature that is shown to be suitable, within the range
3335 °C, for the relevant incubation periods. The temperature should not vary by more than
0.5 °C from the set temperature. The optimal range for pH, multiplicity of infection, cell density,
virus recovery and time of incubation should be established for each manufacturer and should be
approved by the NRA.
The virus suspension should be harvested not later than four days after virus inoculation.
The inoculated cell cultures should be processed in such a
manner that each virus suspension harvested remains
identifiable as a single harvest and is kept separate from other
harvests until the results of all the tests described in Part A
sections A.4.1.2, A.4.1.3, A.4.1.4, A.4.3.3.1, A.4.3.3.2,
A.4.3.3.3, A.4.3.3.4 and A.4.3.3.5 have been obtained.
A 4.3.2 Sampling
Samples required for the testing of single harvests should be taken immediately on harvesting. If
the tests for adventitious agents as described in Part A, section A.4.3.3.3 are not performed
immediately, the samples taken for these tests should be kept at a temperature of 60 °C or lower
and subjected to no more than one freezethaw cycle.
A.4.3.3 Tests on single harvest
A.4.3.3.1 Identity
Each single harvest should be identified as the appropriate poliovirus serotype by immunological
assay on cell culture using specific antibodies or by a molecular method which has been
validated and approved by the NRA.
Page 19
Neutralization tests can distinguish the serotypes of
poliovirus. Molecular method such as sequencing or deep
sequencing, can distinguish Sabin virus from wild-type virus.
Care should be taken to ensure that the sera used are
monospecific by titrating them against homotypic and
heterotypic viruses of known virus titre. Monoclonal
antibodies may be useful in this test.
A.4.3.3.2 Titration for virus content
The virus titre per ml of single harvest should be determined in cell cultures in comparison with
an existing reference preparation (see Appendix 4).
A.4.3.3.3 Tests of neutralized single harvests for adventitious agents
Some selected viruses may be screened by using specific assays such as molecular techniques
(e.g. nucleic acid amplification) (35). For the purposes of the recommendations set out in this
section of Part A, the volume of each single harvest taken for neutralization and testing should be
at least 10 ml and should be such that a total of at least 50 ml or the equivalent of 500 doses of
final vaccine, whichever is the greater, has been withheld from the corresponding single harvest.
The antisera used for neutralization should be of nonhuman origin and should have been
prepared in animals other than monkeys, using virus cultured in cells from a species different
from that used in the production of the vaccine. Samples of each virus harvest should be tested in
human cells and at least one other sensitive cell system.
The neutralized suspensions should be inoculated into bottles of these cell cultures in such a way
that the dilution of the suspension in the nutrient medium does not exceed 1part in 4. The area of
the cell sheet should be at least 3 cm2 per ml of neutralized suspension. At least one bottle of
each kind of cell culture should remain uninoculated and should serve as a control; it should be
maintained using nutrient medium containing the same concentration of the specific antiserum
used for neutralization.
Animal serum may be used in the propagation of the cells,
but the maintenance medium used after inoculation of the test
material should contain no added serum other than the
poliovirus neutralizing antiserum or foetal calf serum of
controlled origin.
The inoculated cultures should be incubated at a temperature of 3537 °C and should be
observed for a period of at least 14 days.
If adequately justified and validated, lower temperatures may
be used.
For the tests to be valid, not more than 20% of the culture vessels should have been discarded for
nonspecific, accidental reasons by the end of the test period.
If any cytopathic changes due to adventitious agents occur in any of the cultures, the virus
harvest should be discarded.
Page 20
New molecular methods with broad detection capabilities are
being developed for detection of adventitious agents. These
methods include degenerate NAT for whole virus families
with analysis of the amplicons by hybridization, sequencing
or mass spectrometry; NAT with random primers followed
by analysis of the amplicons on large oligonucleotide micro-
arrays of conserved viral sequencing or digital subtraction of
expressed sequences; and high throughput sequencing. These
methods might be used in the future to supplement existing
methods or as alternative methods to both in vivo and in vitro
tests after appropriate validation and approval of the NRA
(35).
A.4.3.3.4 Sterility tests for bacteria, fungi and mycoplasmas
A volume of at least 10 ml of each single harvest should be tested for bacterial, fungal, and
mycoplasmal contamination by appropriate tests, as specified in Part A, sections 5.2 (41) and 5.3
(41) of the General requirements for the sterility of biological substances, or by a method
approved by the NRA.
Nucleic acid amplification techniques (NAT) alone or in
combination with cell culture, with an appropriate detection
method, may be used as an alternative to one or both of the
compendial mycoplasma detection methods following
suitable validation and agreement from the NRA (35).
A.4.3.3.5 Test for mycobacteria
The virus harvest should be shown to be free from mycobacteria by an appropriate method
approved by the NRA.
Molecular assays may be used as an alternative to
mycobacteria microbiological culture method tests for the
detection of mycobacteria following suitable validation and
agreement from the NRA (35).
Some manufacturers test for mycobacteria only at the
monovalent bulk stage with the agreement of the NRA
A.4.3.3.6 Tests for molecular consistency of production
Some manufacturers perform a test for the molecular
consistency of production, namely the MAPREC assay (see
section A.4.4.7.1.1) on single harvests. If performed, the
acceptance and rejection criteria of this test should be
updated periodically and approved by the NRA.
A.4.4 Control of monovalent bulk
A.4.4.1 Preparation of monovalent bulk
The monovalent bulk may be prepared by pooling a number of single harvests of the same virus
serotype into a single vessel. This bulk should be filtered through a filter that is able to retain cell
debris.
Page 21
The NRA may require further purification of harvests derived from continuous cell lines. If the
harvests are derived from human diploid or monkey kidney cells, further purification is not
required.
A.4.4.2 Sampling
Samples of the monovalent bulk prepared as described in section A.4.4.1 should be taken
immediately and, if not tested immediately, should be kept at a temperature of -60 °C or below
until the tests described in the following sections are performed.
A.4.4.3 Identity test
Each monovalent bulk should be identified as the appropriate poliovirus serotype by
immunological assay on cell culture using specific antibodies, or by a molecular method which
has been validated and approved by the NRA.
Neutralization tests can distinguish the serotypes of
poliovirus. Molecular methods, such as sequencing or deep
sequencing, can distinguish Sabin virus from wild-type virus.
Care should be taken to ensure that the sera used are
monospecific by titrating them against homotypic and
heterotypic viruses of known virus titre. Monoclonal
antibodies may be useful in this test.
A.4.4.4 Titration for virus content
The virus titre per ml of filtered monovalent bulk should be determined in cell cultures in
comparison with an existing reference preparation (see Appendix 4).
The virus titre as determined by this test should be the basis for the quantity of virus used in the
neurovirulence tests in monkeys or in TgPVR mice (see Part A, section A.4.4.7.2) and for
formulation of the final bulk (see Part A, section A.4.5).
The detailed procedures for carrying out this test and for interpreting the results should be
approved by the NRA.
A.4.4.5 Sterility tests for bacteria and fungi
The final vaccine bulk should be tested for bacterial and fungal sterility as specified in Part A,
section 5.2 of the General requirements for the sterility of biological substances (40).
A.4.4.6 Test for mycobacteria
The virus harvest should be shown to be free from mycobacteria by an appropriate method
approved by the NRA.
Molecular assays may be used as an alternative to
mycobacteria microbiological culture method tests for the
detection of mycobacteria after suitable validation and
agreement from NRA (35).
A.4.4.7 Tests to monitor virus molecular characteristics (consistency)
The poliovirus in the filtered monovalent bulk, prepared as described in section A.4.4.1, should
be tested in comparison with the seed lot or a reference virus preparation (see Part A, section
Page 22
A.1.3) to ensure that the vaccine virus has not undergone changes during its multiplication in the
production cell culture.
A.4.4.7.1 Tests in vitro
The virus in the monovalent bulk should be tested by at least one in vitro test. The test used
should be approved by the NRA. The MAPREC assay provides a sensitive and quantitative
measure for consistency purposes. However, other assays are acceptable after validation. The
assay used historically is to test the property of reproducing virus at temperatures of 36o C and
40o C in comparison with the seed lot or a reference virus preparation of poliovirus of the same
type.
A.4.4.7.1.1 MAPREC
The MAPREC assay is suitable for all three serotypes. Implementation of the assay should be
fully validated by each manufacturer and performed according to the WHO SOP Mutant analysis
by PCR and restriction enzyme cleavage (MAPREC) for oral poliovirus (Sabin) vaccine,
developed from WHO collaborative studies and available from WHO,1 or according to a
validated alternative procedure.
The MAPREC assay should be used to establish the consistency of production once the test has
been validated and normal values for the standards have been established. Depending on a
laboratory’s experience with the MAPREC test, an approach using “warning limits” of ±2
standard deviations and “rejection limits” of ±3 standard deviations may be appropriate.
Acceptance and rejection criteria should be specific to each manufacturer and each working seed
and should be continually updated as each new bulk is prepared. An investigation of consistency
should take place if a batch gives results that are inconsistent with previous production batches.
Results should be expressed as ratios relative to the relevant type-specific International Standard
for MAPREC analysis of poliovirus (Sabin). The acceptable variation of mutant content from
batch to batch should be agreed with the NRA in the light of production and testing experience.
For type 3 (472-C), a batch should be rejected if the level of mutations is above 1.0% when
normalized against the International Standard. The limits for types 1 and 2 should be approved
by the NRA.
Levels of mutations obtained by manufacturers who have
implemented the test for types 1 and 2 virus have been less
than 2.0% for type 1 Sabin (for the sum of both mutations
480-A, 525-C) and 1.5% for type 2 Sabin (481-G) (14).
If a filtered monovalent bulk fails in a MAPREC assay, it cannot be used in the manufacturing of
finished product, and an evaluation of the manufacturing process, including the suitability of the
virus working seed, should be undertaken and discussed with the NRA. Filtered monovalent
bulks that pass the MAPREC assay should be tested subsequently for in vivo neurovirulence.
The assay for type 3 is highly predictive of in vivo
neurovirulence in animal models. No such correlation exists
for types 1 and 2 at the level of revertants present in vaccine
bulks. For these types the assay results provide a measure of
consistency (14).
1 Contact the Coordinator, Quality, Safety and Standards, World Health Organization, 1211, Geneva 27, Switzerland
(http://www.who.int/biologicals/vaccines/en/).
Page 23
Non-radioactive methods for MAPREC are available and
may be introduced after validation and approval by the NRA.
Alternative molecular biology methods that demonstrate an
equivalent or better level of discrimination may be used after
validation and approval by the NRA.
A.4.4.7.1.2 Temperature sensitivity
The monovalent bulk may be tested for the property of reproducing at temperatures of 36 °C and
40 °C in comparison with the seed lot or a reference virus preparation for the marker tests, and
with appropriate rct/40- and rct40+ strains of poliovirus of the same type. The wild-type viruses,
defined as field isolates or reference strains from polioviruses known or believed to have
circulated persistently in the community, which are used as rct40+ controls in this test, should be
contained within the laboratory at progressively higher levels of containment in accordance with
the global action plan and timetable for safe handling of wild polioviruses. The incubation
temperatures used in this test should be controlled to within +/0.1 o C.
The monovalent bulk passes the test if, for both the virus in the monovalent bulk and that in the
appropriate reference material, the titre determined at 36 °C is at least 5.0 log10 greater than that
determined at 40 °C. If the titres obtained for all the reference viruses are not in line with the
expected values, the test should be repeated.
An additional specification that the virus titre must not
exceed 10 CCID50/ml at the higher temperature may also be
applied.
It is desirable that the temperatures used in the test should
also include one in the region of 39.039.5 °C, at which the
titre of the reference material should be reduced by a factor in
the range of 3.05.0 log10 of its value at 36 °C. In one
laboratory, a temperature of 39.2 °C has been found suitable.
It is important to show that the behaviour of the monovalent
bulk is comparable to that of the Sabin reference strain over a
range of temperatures so that a more accurate comparison can
be made.
A.4.4.7.2 Neurovirulence tests
An appropriate in vivo test should be used to evaluate virus seeds and monovalent bulks.
Summaries of the MNVT and TgmNVT, including pass and fail criteria, are given in Appendix 2
along with considerations on the choice of assay.
The test should be approved by the NRA for the specific product and may use transgenic mice or
nonhuman primates or both. The test for neurovirulence in nonhuman primates should be carried
out as summarized in Appendix 2 and described in the SOP Neurovirulence test of types 1, 2 or 3
live attenuated poliomyelitis vaccines (oral) in monkeys, available from WHO.1
1 Contact the Coordinator, Quality, Safety and Standards, World Health Organization, 1211, Geneva 27, Switzerland
(http://www.who.int/biologicals/vaccines/en/).
Page 24
Where the TgmNVT has been approved by the NRA, it should be carried out as summarized in
Appendix 2 and described in detail in the SOP Neurovirulence test of type 1, 2 or 3 live
attenuated poliomyelitis vaccines (oral) in transgenic mice susceptible to poliovirus, available
from WHO ( see above). Its use for batch release purposes should follow the appropriate
validation and implementation processes according to national and international regulations. This
SOP has been validated for vaccines made from Behringwerke SO-derived seeds (types 1 and 2)
and RSO-derived seeds (type 3).
To qualify as competent to perform the TgmNVT test, there is a requirement for laboratories to
complete a standard implementation process as detailed in the SOP. Once qualified as competent,
each laboratory should continue to monitor its performance on a routine basis.
The WHO collaborative study demonstrated that MNVT and TgmNVT are equivalent for testing
vaccines prepared from RSO seeds but that TgmNVT may fail otherwise acceptable (by MNVT)
lots prepared from derivative strains containing additional mutations (28). Therefore, TgmNVT
can be used as a replacement of MNVT for vaccines made from RSO Sabin 3 strain, while the
TgmNVT may require further validation for other derivative strains. This may include the
development of an appropriate homologous reference.
A.4.5 Final bulk
Different final bulks can be formulated.
Final tOPV bulk, final mOPV1 bulk, mOPV3 bulk and final bOPV bulk (bOPV1+3) can be
manufactured using a defined virus concentration of each component.
The operations necessary for preparing the final bulk should be conducted in such a manner as to
avoid contamination of the product.
The dilution and mixing procedures involved in preparing the
final vaccine bulk should be approved by the NRA.
A.4.5.1 Stabilizers
Any stabilizers that may be added to the final bulk should have been shown, to the satisfaction of
the NRA, to improve the stability of the vaccine in the concentrations used and not to impair the
safety of the vaccine.
All the tests described in Part A, sections A.4.3.3 and A.4.4, should be performed on samples
taken before any stabilizers are added.
A.4.5.2 Sterility tests for bacteria and fungi
The final vaccine bulk should be tested for bacterial and fungal sterility, as specified in Part A,
section 5.2 of the General requirements for the sterility of biological substances (40).
A.5 Filling and containers
The requirements concerning filling and containers given in Good manufacturing practices for
biological products (37) should apply to vaccine filled in the final form.
Care should be taken that the material of which the container
is made does not adversely affect the virus content of the
vaccine under the recommended storage conditions.
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A final filtration may be included just before the filling operations.
The manufacturer should provide the NRA with adequate data to prove that the product is stable
under appropriate conditions of storage and shipping
A.6 Control tests on the final lot
Samples should be taken from each filling lot for the tests described in the following sections.
The following tests should be performed on each final lot of vaccine (i.e. in the final containers).
Unless otherwise justified and authorized, the tests should be performed on labelled containers
from each final lot by means of validated methods approved by the NRA. The permissible limits
for the different parameters listed under this section, unless otherwise specified, should be
approved by the NRA.
A.6.1 Inspection of final containers
Every container in each final lot should be inspected visually or mechanically, and those
showing abnormalities should be discarded.
A.6.1.1 Appearance
The appearance of the vaccine should be described with respect to its form and colour.
A.6.2 Extractable volume
Unless otherwise justified and authorized, the extractable volume (ml) and the number of drops,
using the approved dropper, should be determined in a minimum of five individual final
containers.
A.6.3 pH
The pH of the final lot should be tested in a pool of final containers and an appropriate limit set
to guarantee virus stability.
A.6.4 Identity
Each final lot should be identified by immunological assay on cell culture using specific
antibodies or by a molecular method which has been validated and approved by the NRA.
Neutralization tests can distinguish the serotypes of
poliovirus. Molecular methods such as sequencing or deep
sequencing, can distinguish Sabin virus from wild-type virus.
Care should be taken to ensure that the sera used are
monospecific by titrating them against homotypic and
heterotypic viruses of known virus titre. Monoclonal
antibodies may be used for this purpose.
A.6.5 Sterility tests for bacteria and fungi
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Liquid vaccine should be tested for bacterial and fungal sterility, as specified in Part A, section
5.2 of the General requirements for the sterility of biological substances (40), or by the methods
approved by the NRA.
A.6.6 Potency
At least three final containers should be selected at random from each final lot and should be
individually tested in a single assay. The poliovirus content of each serotype, and the total virus
content, should be determined in an assay, as described in Appendix 4 of these recommendations,
using assays that include a reference preparation. When the vaccine contains more than one
poliovirus type, each type should be titrated separately by using appropriate type-specific
antiserum to neutralize each of the other types present. The NRA should specify the minimum
virus titre per human dose.
An internal upper limit may be established by each
manufacturer to monitor the consistency of production (e.g.
based on mean titre CCID50 +3 standard deviations). The
upper limit should be approved by the NRA.
It is recommended that the estimated mean virus titres for a
single human dose of tOPV should be not less than 106.0
CCID50 for type 1, 105.0
CCID50 for type 2, and 105.5
CCID50
for type 3, as determined in an assay described in Appendix 4.
The 95% confidence intervals of the assays should not differ
by a factor of more than 0.3 log10 of the estimated number of
infectious units in the vaccine.
In 1986 the WHO Region of the Americas began to use a
trivalent formulation with 105.8
CCID50 of poliovirus type 3
(44), following a study in Brazil which demonstrated
improved immunogenicity when the amount of type 3 virus
in the trivalent vaccine was increased (45). The subsequent
success in controlling poliomyelitis in the Americas using
this formulation led the EPI Global Advisory Group to
recommend a formulation of trivalent OPV with 106.0
, 105.0
,
105.8
CCID50 per dose for types 1, 2 and 3 respectively, on a
global basis (18, 46).
A.6.7 Thermal stability
Thermal stability should be considered as a vaccine characteristic that provides an indicator of
consistency of production. The thermal stability test is not designed to provide a predictive value
of real-time stability but to evaluate whether the product complies with a defined specification.
Additional guidance on the evaluation of vaccine stability is provided in WHO’s Guidelines on
stability evaluation of vaccines (47).
Three final containers of the vaccine should be incubated at 37 °C for 48 hours. The total virus
content in both exposed and unexposed containers should be determined concurrently with that
of a suitable validated reference preparation. For trivalent vaccines, the vaccine passes the test
when the loss on exposure is not greater than a factor of 0.5 log10 CCID50 per human dose.
Several OPV manufacturers have recently demonstrated that
the thermal stability test specification applied to tOPV
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formulations (loss on exposure is not greater than a factor of
0.5 log10 CCID50 per human dose) is not applicable to some
monovalent and bivalent OPVs. Some manufacturers have
shown that mOPV formulations that failed the current
specification of 0.5 log10 have an acceptable stability profile
throughout the product shelf-life. Therefore, a specification
of 0.6 log10 has been accepted by the NRAs and by the WHO
Prequalification Programme on the basis of documented
evidence that the mOPV1 was stable over two years when
stored at 20 °C or below and six months when stored at
28 °C.
A.6.8 Residual antibiotics (if applicable)
If any antibiotics are added during vaccine production, the content of the residual antibiotics
should be determined and should be within limits approved by the NRA. This test may be
omitted for routine lot release once consistency of production has been established to the
satisfaction of the NRA.
A.6.9 Stabilizer (if applicable)
If a stabilizer is added during vaccine production, the content of the stabilizer should be
determined and should be within limits approved by the NRA.
A.7 Records
The recommendations given in section 8 of Good manufacturing practices for biological
products (37) should apply.
A.8 Retained samples
The requirements given in section 9.5 of Good manufacturing practices for biological products
(37) should apply.
A.9 Labelling
The requirements given in section 7 of Good manufacturing practices for biological products
(37) should apply, with the addition of the following.
The label on the container or package should include the following information:
the designation(s) of the strain(s) of poliovirus contained in the vaccine;
the minimum amount of virus of each type contained in one recommended human
dose;
the cell substrate used for the preparation of the vaccine, and the nature and
amount of any stabilizer present in the vaccine;
a statement that the vaccine is not to be injected;
the number of doses in each vial;
the volume of the dose.
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It is desirable for the label to carry the names both of the
producer and of the source of the bulk material if the
producer of the final vaccine did not prepare it. The nature
and amount of the antibiotics present in the vaccine, if any,
may be included.
A.10 Distribution and shipping
The requirements given in section 8 of Good manufacturing practices for biological products
(37) should apply. Further guidance is provided in WHO’s Model guidance for the storage and
transport of time- and temperature-sensitive pharmaceutical products (48).
A.11 Stability, storage and expiry date
A.11.1 Stability testing
Adequate stability studies form an essential part of vaccine development. Current guidance on
the evaluation of vaccine stability is provided in WHO’s Guidelines on stability evaluation of
vaccines (47). Stability testing should be performed at different stages of production, namely on
single harvests, monovalent bulk, final bulk and final lot. Stability-indicating parameters should
be defined or selected appropriately according to the stage of production. A shelf-life should be
assigned to all in-process materials during vaccine production – particularly intermediates such
as single harvests, monovalent bulk and final bulk.
The stability of the vaccine in its final container and at the recommended storage temperatures
should be demonstrated to the satisfaction of the NRA on at least three consecutive lots of final
product. Accelerated thermal stability tests may be undertaken to give additional information on
the overall characteristics of a vaccine.
The formulation of vaccine should be stable throughout its shelf-life. Acceptable limits for
stability should be agreed with NRAs. Following licensure, ongoing monitoring of vaccine
stability is recommended to support shelf-life specifications and to refine the stability profile
(47). Data should be provided to the NRA in accordance with local regulatory requirements.
Where vaccine is to be stockpiled, manufacturers should conduct real-time stability studies on
monovalent bulks at 40 °C or below, or on finished monovalent, bivalent and trivalent
composition at 20 °C.
Any extension of the shelf-life should be approved by the NRA.
The final stability testing programme should be approved by the NRA and should include an
agreed set of stability-indicating parameters, procedures for the ongoing collection and sharing
of stability data, and criteria for the rejection of vaccine(s).
A.11.2 Storage conditions
Before being released by the manufacturing establishment, all vaccines in final containers should
be kept continuously in the frozen state at a temperature below 20 °C.
The manufacturer should indicate conditions of storage and shipping that will ensure that the
vaccine conforms to the requirements of potency until the expiry date stated on the label. These
must be approved by the NRA.
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Although the recommended storage temperature is at 20 ºC, vaccine may be stored at 28ºC for
six months. During shipment or in the field, the vaccine may be thawed and refrozen.
Manufacturers should demonstrate that multiple freezethaw
cycles do not adversely affect the quality of the product. The
number of freezethaw cycles should be approved by the
NRA.
The total storage period at 28 °C should not exceed six months. Stability data should be
generated for each formulation of OPV to support these excursions in the storage conditions at
28 °C following thawing, and this should be approved by the NRA.
A.11.3 Expiry date
The expiry date should be defined on the basis of the shelf-life and should be supported by the
stability studies with the approval of the NRA. The expiry date should relate to the date of filling
or the date of the first valid titration for virus content after filling, performed in an assay of virus
concentration as described in Appendix 4 (i.e. the date of the potency test).
The label should specify only one storage temperature and expiry date.
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Part B. Nonclinical evaluation of live attenuated poliomyelitis vaccines (oral)
The nonclinical evaluation of candidate live attenuated poliomyelitis vaccines (oral) should be
based on WHO guidelines on nonclinical evaluation of vaccines (15). The following specific
issues should be considered in addition to the tests described in sections A.3.2.3 and A.3.2.4 in
the context of a change in virus seed or manufacturing process for OPV.
B.1 Characterization of a new virus sub-master seed from the WHO master seed
In the event that a new virus sub-master seed is prepared by a single passage from the WHO
master seed, it should be subjected to extensive characterization which should include evaluation
of the virus working seeds and at least three monovalent bulks derived from it, as described in
section A.4.4.7. Characterization studies must include the evaluation of identity by complete
nucleotide sequencing to prove that the new sub-master seed consensus sequence is identical to
conventional Sabin master seeds and that the mutational composition (e.g. in MAPREC) is
consistent. Massively parallel sequencing may also be undertaken to determine the distribution
of mutants. These approaches have not yet been formally validated, other than the MAPREC
tests for base positions in the 5′ noncoding region, as described in section A.4.4.7.1.1. A new
sub-master seed should be tested for neurovirulence in the MNVT or the TgmNVT. Summaries
of the MNVT and TgmNVT are given in Appendix 2 along with considerations on the choice of
assay.
B.2 Characterization of virus working seeds from an established master seed where passage level between master and working seed is increased
The acceptable passage level of live polio vaccines relative to the original seeds is rigidly
specified as there is evidence for some seeds that increases in virulence have occurred with
increasing passage. However, due to the limited stocks of master seeds available, it may be
necessary in the future for some manufacturers to prepare working seed lots by expanding
current seed lots with an additional passage. The new virus working seed lots will require careful
comparative studies with the previously approved working seed lot and will need to meet the
criteria outlined in sections A.3.2.3 and A.3.2.4. At least three monovalent bulks produced from
the new virus working seed lot should also be tested and shown to meet the requirements of
section A.4.4.7.
B.3 Characterization following changes in the manufacturing process
If the OPV manufacturing process is new or major changes are implemented in vaccine
production – such as changing from primary monkey cells to cell lines – extensive assessment
should be conducted to ensure that the mutational composition is not significantly altered by the
new manufacturing process. This evaluation may include nucleotide sequencing and studies of
mutant accumulation during passage in production cultures by using MAPREC and other
molecular methods such as massively parallel sequencing. The new virus working seed lots will
meet the criteria outlined in sections A.3.2.3 and A.3.2.4. At least three monovalent bulks
Page 31
produced from it also need to be tested and shown to meet the requirements of section A.4.4.7. In
addition, based on the results of the genetic characterization and the animal neurovirulence tests,
clinical studies may be required (see Part C).
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Part C. Clinical evaluation of live attenuated poliomyelitis vaccines (oral)
Clinical trials should adhere to the principles described in WHO’s Guidelines for good clinical
practice (GCP) for trials on pharmaceutical products (49) and Guidelines on clinical evaluation
of vaccines: regulatory expectations (16). All clinical trials should be approved by the relevant
NRAs.
Some of the issues that are specific to the clinical evaluation of OPVs are discussed in the
following sections. These sections should be read in conjunction with the general guidance
mentioned above. It is also recommended that manufacturers should consult with relevant NRAs
regarding the overall clinical development programme.
Part C considers the provision of clinical data required for:
new formulations based on licensed OPV that is based on Sabin poliovirus strains,
including monovalent, bivalent and trivalent vaccines;
situations where there have been major changes to the manufacturing process of
an established vaccine (e.g. changing from primary monkey kidney cells to a cell
line).
Clinical evaluation is not required for a vaccine manufactured by using a new virus working seed
lot provided that the passage level is not more than one from the master seed lot, the working
seed has been characterized, and consistency of the manufacturing process has been
demonstrated (see sections A.3.2.3 and A.3.2.4). Generation of a new sub-master seed will
require extensive characterization but not clinical trials (see Part B).
Vaccine formulations containing one or two poliovirus serotypes have been licensed on the basis
of clinical trials in endemic countries. The results of clinical trials in Egypt and northern India
have demonstrated that the efficacy of mOPV1 is superior to that of trivalent OPV in terms of
inducing immunity against poliovirus type 1 (18, 50). Health authorities have recommended
widespread use of this vaccine to eliminate poliovirus type 1 transmission in India. In addition,
studies on bOPV containing type 1 and type 3 have demonstrated that bOPV is “non-inferior” to
mOPV1 and mOPV3 individually, and “superior” to tOPV. Therefore, based on these results, the
Advisory Committee on Poliomyelitis Eradication (ACPE) recommended that bOPV should be
used to complement tOPV in routine immunization and to complement tOPV and mOPVs in
supplementary immunization activities.
C.1 General considerations
The poliomyelitis eradication initiative which followed World Health Assembly resolution
WHA41.28 of 1988 has led to a dramatic decrease in poliomyelitis cases globally (17).
Therefore, efficacy studies for poliovirus vaccines are not feasible, and clinical evaluation and
seroprevalence studies should be based on comparative assessment of the safety and
immunogenicity of new candidate vaccines with a licensed vaccine (comparator vaccine). The
assessment of seroconversion should be based on the elicitation of neutralizing antibodies, which
are the basis of protection (17). The approval of a new candidate OPV should be based on a clear
demonstration of non-inferiority compared to currently licensed OPV. The relative risk of VAPP
Page 33
for a new candidate vaccine versus approved vaccines cannot be estimated from pre-approval
studies but should be addressed as part of post-marketing surveillance.
C.2 Safety and immunogenicity studies
C.2.1 Assessment of the immune response
A serum neutralizing antibody titre of 1/4–1/8 is considered to be a marker of protection against
poliovirus (51). The demonstration of an immune response to OPV vaccination should be based
on the measurement of neutralizing antibody titres pre- and post-vaccination. Geometric mean
titres (GMTs), seroconversion rates and reverse cumulative distributions should be provided.
Seroconversion for polio antigen is defined as:
for subjects seronegative at the pre-vaccination time point: antibody titres above
the cut-off (titre 1/41/8);
for subjects seropositive at the pre-vaccination timepoint: antibody titres that are
fourfold the expected titre of maternal antibodies based on the pre-vaccination
titre declining with a half-life of 28 days;
in populations with high antibody titres: a change from below the highest dilution
tested (<8192) to above the highest dilution tested (>8192) will also indicate
seroconversion.
The half-life of maternal antibody decay may be assumed to be 28 days (52). It is desirable to
consider these two parameters separately in the comparison between a new OPV and a licensed
one used as control.
WHO has made an effort to standardize polio virology methods, leading to the publication in
1990 of WHO’s Manual for the virological investigation of polio (53). It is recommended that a
standardized technique for measurement of neutralizing antibodies, involving standard cell lines
and other standard reagents, should be used, such as International Standards of anti-poliovirus
sera for types 1, 2 and 3, and that the results should be expressed in international units of
neutralizing antibody (54, 55).
C.2.2 Immunogenicity studies
New candidate OPVs manufactured using different vaccine compositions (e.g. monovalent or
bivalent) should be compared with a licensed formulation. New candidate vaccines should be
compared to at least one well-established and licensed OPV. The comparator vaccine(s) selected
should have been in use for some years so that some data on effectiveness are available in
addition to a reliable description of the safety profile.
C.2.3 Population
The evaluation of new OPV formulations, including monovalent, bivalent and trivalent vaccines,
based on Sabin strains may be conducted in infants and newborns since safety profiles in these
populations have already been established.
The study exclusion criteria should reflect the current contraindications to administration of
OPVs.
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C.2.4 Endpoints and analyses
The clinical study protocol should state the primary objective(s) of the study. The neutralizing
antibody response to the candidate vaccine should be demonstrated to be non-inferior versus an
appropriate licensed OPV based primarily on GMTs and/or seroconversion rates. The primary
endpoint should be selected according to the study population and the anticipated immune
response. For example, very high seroprevalence rates are expected in highly immunized
populations, thus having implications for the selection of the non-inferiority margin and
therefore the sample size calculation. Further details on demonstrating non-inferiority are
described in the WHO Guidelines on clinical evaluation of vaccines: regulatory expectations
(16).
Other immunological parameters should be compared in planned secondary analyses (e.g.
percentages reaching predefined titres).
C.2.5 Dose-ranging studies
At the time of preparation of this document, all licensed OPV formulations (e.g. monovalent,
bivalent and trivalent) contained the recommended dose for each poliovirus type (not less than
106.0
CCID50 for type 1, 105.0
CCID50 for type 2, and 105.5
CCID50 for type 3). However,
development of novel formulations with improved stability (addition of stabilizers/excipients) or
immunogenicity (used in combination with an adjuvant) may require dose-ranging studies to
determine the minimum dose of virus required in CCID50 to provide adequate immune responses
(16). These data could also be used to support the derivation of the minimum viral titre that
should be present in the vaccine at the end of its shelf-life.
C.2.6 Vaccine virus shedding and transmission
Changes in vaccine composition may impact virus replication in the intestinal tract and may
influence the ability to induce an immune response. Manufacturers should undertake studies to
determine the profile of the vaccine virus (if applicable, by serotype) excreted in the stools of
vaccinees and the duration of shedding. Evaluation of virus excretion of new vaccine
formulations containing one, two or three serotypes (i.e. monovalent, bivalent or trivalent)
should be evaluated in comparison to the trivalent licensed formulation (18).
C.2.7 Challenge studies with attenuated Sabin poliovirus
Induction of mucosal immunity by the candidate and the comparator vaccines should be
determined by the assessment of virus excretion after the administration of a challenge dose of
mOPV. Excretion of poliovirus in stool specimens is determined at various intervals immediately
before the challenge (day 0) and on days 7, 14, 21 and 28 thereafter (50).
C.2.8 Concomitant administration with other vaccines
An evaluation of the effects of co-administration of an OPV with other vaccines should be
considered, taking into account which vaccines are most likely to be given concomitantly in
different age groups and populations.
When OPVs are used in an EPI programme simultaneously with other vaccines, it is particularly
important that the effects of co-administration should be evaluated (e.g. co-administration studies
with rotavirus vaccines which are also administered by the oral route).
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Immune responses to all other antigens co-administered with the new OPV should be measured
at least in subsets. While the study will usually be powered only to demonstrate non-inferiority
with respect to neutralizing antibody against the different poliovirus types used in the vaccine,
the protocols should at least include planned secondary analyses of antigen-specific responses. If
these analyses indicate that immune responses are lower on co-administration with a new OPV
compared to the licensed vaccine(s), NRAs will need to consider the potential clinical
consequences on a case-by-case basis.
C.2.9 Pre-licensure safety data
The general approach to the assessment of safety of a new OPV during clinical studies should be
in accordance with WHO’s Guidelines on clinical evaluation of vaccines: regulatory
expectations (16). Planned safety studies should be supported by a clear scientific rationale.
Given the long history of the use of vaccines based on Sabin strains, the NRA may decide that
additional pre-licensure safety studies are not required. Where a new vaccine formulation, which
has not been used previously, is investigated, larger-scale studies will be needed.
An appropriate pharmacovigilance plan should be developed and approved by the NRA prior to
licensure.
C.3 Post-marketing studies and surveillance
Enhanced safety surveillance, particularly for detection of VAPP, should be undertaken during
the initial post-approval years in collaboration with NRAs. Manufacturers and health authorities
should work in collaboration with the global polio surveillance laboratory network to monitor
new vaccines once they are introduced into immunization programmes. These laboratories have
extensive experience in poliovirus surveillance and may provide excellent surveillance and post-
marketing support.
The total duration of enhanced surveillance should be regularly reviewed by the NRA. If
particular issues arise during pre-licensure studies or during post-licensure safety surveillance, it
may be necessary to conduct specific post-licensure safety studies.
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Part D. Recommendations for national regulatory authorities
D.1 General
The general recommendations for national regulatory authorities and national control
laboratories given in the Guidelines for national authorities on quality assurance for biological
products (56) and Guidelines for independent lot release of vaccines by regulatory authorities
(57) should apply.
The detailed production and control procedures, as well as any significant changes in them that
may affect the quality, safety and efficacy of live attenuated OPV should be discussed with and
approved by the NRA.
For control purposes, the International Standards currently in force should be obtained for the
purpose of calibration of the national/regional/working standards (58). The NRA may obtain the
product-specific/working reference from the manufacturer to be used for lot release until the
international/national standard preparation is established.
Only a monovalent bulk approved by the NRA with regard to the neurovirulence test can be used
by the manufacturer for the formulation of a final bulk.
If the national control laboratory does not perform the monkey neurovirulence test itself, it
should carry out a second reading of the histological sections provided by the manufacturer for
each monovalent bulk. In addition, the national control laboratory should perform a second
reading of at least four neurovirulence tests on the reference preparations using the monkey
neurovirulence test in order to obtain the necessary baseline data for comparison with the
neurovirulence of test vaccines.
The national control laboratory should encourage the use of the standard form for the reporting
of data on virus activity in the sections taken from histopathological examination.
If the national control laboratory performs the mouse neurovirulence test itself, it should
complete the standard implementation process.
If the national control laboratory does not perform the transgenic mouse neurovirulence test, it
should carry out a clinical scoring of mice in parallel with the manufacturer at least at days 3 and
4 plus day 14 for each monovalent bulk. Moreover, once a year, the injection of mice should be
observed by the national control laboratory. Only appropriately trained staff from a competent
national control laboratory can carry out a clinical scoring of mice in parallel with the
manufacturer.
In one region, 1 in 10 bulks is also independently tested by a
national control laboratory competent in carrying out the test.
Other regions that implement the transgenic mouse
neurovirulence test may wish to follow this approach.
Consistency of production has been recognized as an essential component in the quality
assurance of live attenuated OPV. In particular, the NRA should carefully monitor production
Page 37
records and quality control test results for clinical lots, as well as a series of consecutive lots of
the vaccine.
D.2 Official release and certification
A vaccine lot should be released only if it fulfils the national requirements and/or satisfies Part A
of the present recommendations (57).
A protocol based on the model given in Appendix 5, signed by the responsible official of the
manufacturing establishment, should be prepared and submitted to the NRA in support of a
request for release of the vaccine for use.
A statement signed by the appropriate official of the NRA should be provided to the
manufacturing establishment and should certify that the lot of vaccine in question meets all
national requirements, as well as Part A of these recommendations. The certificate should
provide sufficient information on the vaccine lot. A model certificate is given in Appendix 6.
The official national release certificate should be provided to importers of the vaccines. The
purpose of the certificate is to facilitate the exchange of vaccines between countries.
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Part E. Recommendations for live attenuated poliomyelitis vaccine (oral) prepared in primary cultures of monkey kidney
The following additional or alternative recommendations are for OPV prepared in cultures of
primary monkey kidney cells and concern the testing of the cell substrate used for the production
of the vaccine (Part A, section A.4). They should therefore be added to, or substituted for, the
appropriate sections in Part A. Recommendations E.4.1, E.4.3.1.1, E.4.4.1 and E.4.4.2.1 are
additions; recommendations E.4.2.1 and E.4.2.2 together replace A.4.2.1, and E.4.2.3 replaces
A.4.1. All the other recommendations given in Parts A and B of the document are also applicable
to this vaccine.
E.4 Control of vaccine production
E.4.1 Control of source materials
E.4.1.1 Monkeys used for preparation of kidney-cell cultures and for testing of virus.
If vaccine is prepared in monkey kidney-cell cultures, animals of a species approved by the NRA,
in good health and not previously employed for experimental purposes, should be used.
Manufacturers should use animals from closed or intensively monitored colonies.
The monkeys should be kept in well-constructed and adequately ventilated animal rooms in
cages separated in such a way as to prevent cross-infection between cages, together with other
adequate precautionary measures. Cage-mates should not be interchanged. The monkeys should
be kept in the country of manufacture of the vaccine in quarantine groups1 for a period of not
less than six weeks before use. If at any time during the quarantine period the overall death rate
of a shipment consisting of one or more groups reaches 5% (excluding deaths from accidents or
where the cause was specifically determined not to be an infectious disease), monkeys from that
entire shipment should continue in quarantine for a further period of not less than six weeks. The
monkeys used should be free of infection. At the end of the extended quarantine period, and
following thorough investigations, if any additional monkeys die of the same infectious disease,
the entire group is discarded from production.
The groups should be kept continuously in isolation, as in quarantine, even after completion of
the quarantine period, until the monkeys are used. After the last monkey of a group has been
taken, the room that housed the group should be thoroughly cleaned and decontaminated before
being used for a fresh group.
In countries in which the kidneys from near-term monkeys
are used, the mother should be quarantined for the term of
pregnancy.
All actions taken by working personnel should be based on the assumption that a great potential
hazard exists at all times in the quarantine area. Personnel should be provided with protective
clothing, including gloves, footwear and masks or visors. Street clothes should not be permitted
1 A quarantine group is a colony of selected healthy monkeys kept in one room, with separated feeding and cleaning
facilities, and having no contact with other monkeys during the quarantine period.
Page 39
in the animal rooms. Smoking, eating and drinking should be forbidden while personnel are in
the animal rooms.
A supervisor should be made responsible for reporting unusual illness among employees and for
ensuring that all injuries are properly treated. No worker who has cuts or abrasions on exposed
areas of the body should enter the animal area. Any unexplained febrile illness, even while off
duty, should be considered as potentially related to the employee’s occupation.
Monkeys from which kidneys are to be removed should be anaesthetized and thoroughly
examined, particularly for evidence of tuberculosis and herpes B virus infection.
If a monkey shows any pathological lesion relevant to the use of its kidneys in the preparation of
a seed lot or vaccine, it should not be used, nor should any of the remaining monkeys of the
quarantine group concerned be used unless it is evident that their use will not impair the safety of
the product.
All the operations described in this section should be conducted outside the areas where vaccine
is made.
The monkeys should be shown to be free from antibodies to SV40 virus and simian
immunodeficiency virus.
It is desirable that kidney-cell cultures are derived from
monkeys shown to be free from antibodies to foamy viruses.
In some countries, monkeys are tested for antibodies to
herpes B virus.
E.4.2 Production precautions
The general production precautions called for by the Good manufacturing practices for
biological products (37) should apply to the manufacture of vaccine, with the addition of the
following tests.
E.4.2.1 Monkey kidney-cell cultures for vaccine production
Cultures of monkey kidney cells should be prepared from kidneys that have shown no
pathological signs. Virus for the preparation of vaccine should be grown by aseptic methods in
such cultures. If animal serum is used in the propagation of the cells, the maintenance medium
used after virus inoculation should contain no added serum.
To reduce animal use, the virus may be grown in serially
passaged monkey kidney-cell cultures from primary monkey
kidney cells.
Each group of cell cultures derived from a single monkey, or from no more than 10 near-term
monkeys, should be prepared and tested as an individual group.
E.4.2.2 Tests of cell cultures used for vaccine production (see Appendix 7)
On the day of inoculation with virus working seed lot, each cell culture should be examined for
degeneration caused by an infective agent. If, in this examination, evidence is found of the
presence in a cell culture of any adventitious agent, the entire group of cultures concerned should
not be used for vaccine production.
Page 40
On the day of inoculation with the virus working seed lot, a sample of at least 30 ml of the
pooled fluid removed from the cell cultures of the kidneys of each single monkey, or from no
more than 10 near-term monkeys, should be divided into two equal portions. One portion of the
pooled fluid should be tested in monkey kidney-cell cultures prepared from the same species, but
not the same animal, as that used for vaccine production. The other portion of the pooled fluid
should be tested in kidney-cell cultures from another species of monkey, provided that the tests
are done in cell cultures from at least one species known to be sensitive to SV40 virus. The
pooled fluid should be inoculated into bottles of these cell cultures in such a way that the dilution
of the pooled fluid in the nutrient medium does not exceed 1 part in 4. The area of the cell sheet
should be at least 3 cm2 per ml of pooled fluid. At least one bottle of each kind of cell culture
should remain uninoculated and should serve as a control.
When the monkey species used for vaccine production is
known to be sensitive to SV40 virus, a test in a second
species may be omitted with the approval of the NRA.
Animal serum may be used in the propagation of the cells,
provided that it does not contain SV40 antibody or other
inhibitors, but the maintenance medium used after
inoculation of the test material should contain no added
serum except as described below.
The cultures should be incubated at a temperature of 3537 °C and should be observed for a total
period of at least four weeks. During this observation period, and after not less than two weeks’
incubation, from each of these cultures at least one subculture of fluid should be made in the
same tissue culture system. The subculture should also be observed for at least two weeks.
Serum may be added to the original culture at the time of
subculturing, provided that the serum does not contain SV40
antibody or other inhibitors. Immunochemical techniques
may be useful for detecting SV40 and other viruses in the
cells.
A further sample of at least l0 ml of the pooled fluid should be tested for the presence of herpes
B virus and other viruses in rabbit kidney-cell cultures. Serum used in the nutrient medium of
these cultures should have been shown to be free from inhibitors.1 The sample should be
inoculated into bottles of these cell cultures in such a way that the dilution of the pooled fluid in
the nutrient medium does not exceed 1 part in 4. The area of the cell sheet should be at least 3
cm2 per ml of pooled fluid. At least one bottle of the cell cultures should remain uninoculated
and should serve as a control.
The cultures should be incubated at a temperature of 3537 °C and should be observed for a
period of at least two weeks.
It is suggested that, in addition to these tests, a further sample
of l0 ml of pooled fluid removed from the cell cultures on the
day of inoculation with the seed lot virus should be tested for
the presence of adventitious agents by inoculation into cell
cultures sensitive to measles virus.
1 Human herpesvirus (herpes simplex) has been used as an indicator for freedom from B virus inhibitors because of
the danger of handling herpes B virus.
Page 41
For the tests to be valid, not more than 20% of the culture vessels should have been discarded for
nonspecific, accidental reasons by the end of the respective test periods.
If, in these tests, evidence is found of the presence of an adventitious agent, the single harvest
from the whole group of cell cultures concerned should not be used for vaccine production.
If the presence of the herpes B virus is demonstrated, the manufacture of vaccine should be
discontinued and the NRA should be informed. Manufacturing should not be resumed until a
thorough investigation has been completed and precautions have been taken against any
reappearance of the infection, and then only with the approval of the NRA.
If these tests are not carried out immediately, the samples of pooled cell-culture fluid should be
kept at a temperature of 60 °C or below, with the exception of the sample for the test for B
virus, which may be held at 4 °C provided that the test is done not more than seven days after the
sample has been taken.
E.4.2.3 Test of control cell cultures
Cultures prepared on the day of inoculation with the virus working seed lot from 25%, but not
more than 2.5 litres, of the cell suspension obtained from the kidneys of each single monkey, or
from not more than 10 near-term monkeys, should remain uninoculated and should serve as
controls. These control cell cultures should be incubated under the same conditions as the
inoculated cultures for at least two weeks, and should be examined during this period for
evidence of cytopathic changes. For the tests to be valid, not more than 20% of the control cell
cultures should have been discarded for nonspecific, accidental reasons. At the end of the
observation period, the control cell cultures should be examined for degeneration caused by an
infectious agent. If this examination, or any of the tests required in this section, shows evidence
of the presence in a control culture of any adventitious agent, the poliovirus grown in the
corresponding inoculated cultures from the same group should not be used for vaccine
production.
E.4.2.3.1 Tests for haemadsorbing viruses
At the time of harvest, or not more than four days after the day of inoculation of the production
cultures with the virus working seed lot, a sample of 4% of the control cell cultures should be
taken and should be tested for haemadsorbing viruses. At the end of the observation period, the
remaining control cell cultures should be similarly tested. The tests should be carried out as
described in Part A, section A.4.1.2.
E.4.2.3.2 Tests for other adventitious agents
At the time of harvest, or no more than seven days after the day of inoculation of the production
cultures with the virus working seed lot, a sample of at least 20 ml of the pooled fluid from each
group of control cultures should be taken and tested in two kinds of monkey kidney-cell culture,
as described in Part E, section E.4.2.2.
At the end of the observation period for the original control cell cultures, similar samples of the
pooled fluid should be taken and the tests referred to in this section in the two kinds of monkey
kidney-cell culture and in the rabbit-cell culture should be repeated, as described in Part E,
section E.4.2.2.
Page 42
If the presence of herpes B virus is demonstrated, the production cell cultures should not be used
and the measures concerning vaccine production described in Part E, section E.4.2.2, should be
taken.
In some countries, fluids are collected from the control cell
cultures at the time of virus harvest and at the end of the
observation period. Such fluids may then be pooled before
testing for adventitious agents.
E.4.3 Control of single harvests
E.4.3. 1 Single harvest
E.4.3.1.1 Tests for neutralized single harvests in monkey kidney-cell cultures
A sample of at least 10 ml of each single harvest should be neutralized by type-specific
poliomyelitis antiserum prepared in animals other than monkeys. In preparing antisera for this
purpose, the immunizing antigens used should be prepared in non-simian cells.
Care should be taken to ensure that the antiserum used is
monospecific. This may be demonstrated by titration of the
antiserum against homotypic and heterotypic virus of known
virus titre using the same dilution of the antiserum as that
used for neutralization.
Half (corresponding to at least 5 m1 of single harvest) of the neutralized suspension should be
tested in monkey kidney-cell cultures prepared from the same species, but not the same animal,
as that used for vaccine production. The other half of the neutralized suspension should be tested
in monkey kidney-cell cultures from another species, provided that the tests are done in cell
cultures from at least one species known to be sensitive to SV40 virus.
The neutralized suspensions should be inoculated into bottles of these cell cultures in such a way
that the dilution of the suspension in the nutrient medium does not exceed 1 part in 4. The area of
the cell sheet should be at least 3 cm2 per ml of neutralized suspension. At least one bottle of
each kind of cell culture should remain uninoculated to serve as a control and should be
maintained using nutrient medium containing the same concentration of the specific antiserum
used for neutralization.
Animal serum may be used in the propagation of the cells
provided that it does not contain inhibitors, but the
maintenance medium used after the inoculation of the test
material should contain no added serum other than the
poliovirus neutralizing antiserum, except as described below.
The cultures should be incubated at a temperature of 3537 °C and should be observed for a total
period of at least four weeks. During this observation period, and after no less than two weeks’
incubation, at least one subculture of fluid should be made from each of these cultures in the
same tissue culture system. The subcultures should also be observed for at least two weeks.
Serum may be added to the original cultures at the time of
subculturing provided that the serum does not contain
inhibitors. Immunohistochemical techniques may be useful
for detecting SV40 and other viruses in the cells.
Page 43
It is suggested that, in addition to these tests, a further sample
of the neutralized single harvest is tested by inoculation of 10
ml into human cell cultures sensitive to measles virus.
For the tests to be valid, not more than 20% of the culture vessels should have been discarded for
nonspecific, accidental reasons by the end of the respective test periods.
If any cytopathic changes occur in any of the cultures, the causes of these changes should be
investigated. If the cytopathic changes are shown to be due to unneutralized poliovirus, the test
should be repeated. If there is evidence of the presence of SV40 virus or other adventitious
agents attributable to the single harvest, that single harvest should not be used for vaccine
production.
E.4.4 Control of monovalent bulk
E.4.4.1 Monovalent bulk (before filtration)
E.4.4.1.1 Tests in rabbits
A sample of the monovalent bulk should be tested for the presence of herpes B virus and other
viruses by injection in at least 10 healthy rabbits each weighing between 1.5 and 2.5 kg. The
sample should consist of at least 100 ml. Each rabbit should receive not less than 10 ml and not
more than 20 ml, of which 1 ml is given intradermally at multiple sites, and the remainder
subcutaneously. The rabbits should be observed for between three and five weeks for death or
signs of illness.
It is suggested that the sample should consist of at least 1% of
monovalent bulk, provided that this is not less than 100 ml,
up to a maximum of 500 ml.
All rabbits that die after the first 24 hours of the test should be examined by autopsy, the brain
and organs being removed for detailed examination to establish the cause of death. Animals
showing signs of illness should be humanely killed and subjected to a similar autopsy.
The monovalent bulk passes the test if no more than 20% of the inoculated rabbits show signs of
intercurrent infection during the observation period and if none of the rabbits shows evidence of
infection with B virus or other adventitious agents or lesions of any kind attributable to the bulk
suspension.
If the presence of B virus is demonstrated, the measures concerning vaccine production
described in Part E, section E.4.2.2, should be taken.
A test for the presence of Marburg virus may be carried out
in guinea pigs.
E.4.4.2 Monovalent bulk (after filtration)
E.4.4.2.1 Tests for retroviruses
Test samples from the filtered monovalent bulk should be examined for the presence of
retroviruses by an assay for reverse transcriptase (RTase) acceptable to the NRA (36).
Page 44
Authors and acknowledgements
The first draft of this document was prepared by Dr M. Ferguson, Horning, United Kingdom;
Dr P. Minor, National Institute for Biological Standards and Control, Potters Bar, United
Kingdom; Dr K. Chumakov, Center for Biologics Evaluation and Research, Food and Drug
Administration, Rockville, MD, USA; Dr M. Baca-Estrada, Health Canada, Ottawa, Canada; Ms
V. Pithon, Agence Française de Sécurité Sanitaire des Produits de Santé, Lyon, France; with
support from the World Health Organization secretariat: Dr J. Fournier-Caruana, Dr I. Knezevic,
Dr D.J. Wood, Dr T.Q. Zhou (Immunization, Vaccines and Biologicals), and Dr R. Sutter (Polio
Eradication Initiative), World Health Organization, Geneva, Switzerland; taking into
considerations the discussions at a working group meeting on “Technical specifications for
manufacturing and evaluating the WHO recommendations for OPV: TRS Nos. 904 and 910”,
held in Geneva, Switzerland 2022 July 2010 and attended by (alphabetical by country):
Dr G. Waeterloos, Scientific Institute of Public Health, Brussels, Belgium; Dr I. Ernest and Dr B.
Descambe, GSK Biologicals, Belgium (representing the International Federation of
Pharmaceutical Manufacturers and Associations); Ms V. Pithon, Agence Francaise de Sécurité
Sanitaire de Produits de Santé, France; Ms E. Coppens, Sanofi Pasteur, France (representing the
International Federation of Pharmaceutical Manufacturers & Associations); Dr C. Milne,
European Directorate for the Quality of Medicines and Health Care, Council of Europe, France;
Ms I.S. Budiharto and Mr A. Azhari, Bio Farma, Bandung, Indonesia (representing the
Developing Countries Vaccine Manufacturing Network); Dra L. Herawati and Dra D. Kusmiaty,
National Agency of Drug and Food Control, Jakarta, Indonesia; Dr L. Fiore and Dr A.L. Salvati,
Istituto Superiore di Sanità, Rome, Italy; Mr D. Mattii and Mr T. Pasquali, Novartis Vaccines
and Diagnostics, Italy (representing the International Federation of Pharmaceutical
Manufacturers and Associations); Prof A. Nomoto, Microbial Chemistry Research Foundation,
Tokyo, Japan; Dr R.C. Rosales and Dr J. B. González, Birmex, Mexico (representing the
Developing Countries Vaccine Manufacturing Network); Dr L.Y. Yuan, National Institute for
the Control of Pharmaceutical and Biological Products, Beijing, China; Dr P. Minor, Dr J.
Martin, Dr A. MacAdam, Dr G. Dunn and Dr A. Heath, National Institute for Biological
Standards and Control, Potters Bar, United Kingdom; Dr E. Dragunsky and Dr K. Chumakov,
Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD,
USA; Dr E. Wimmer, State University of New York, New York, NY, USA; Dr N. Nathanson,
University of Pennsylvania, USA; with support from the World Health Organization secretariat:
Dr M. Baca-Estrada, Dr C. Conrad, Dr J. Fournier-Caruana, Dr I. Knezevic, Dr D.J. Wood and
Dr T.Q. Zhou (Immunization, Vaccines and Biologicals), and Dr R. Sutter (Polio Eradication
Initiative), World Health Organization, Geneva, Switzerland.
The second draft was prepared following a meeting of the drafting group attended by Dr M.
Ferguson, Horning, United Kingdom; Dr P. Minor, National Institute for Biological Standards
and Control, Potters Bar, United Kingdom; Dr K. Chumakov, Center for Biologics Evaluation
and Research, Food and Drug Administration, Rockville, MD, USA; Dr M. Baca-Estrada, Health
Canada, Ottawa, Canada; Ms V. Pithon, Agence Française de Sécurité Sanitaire des Produits de
Santé , Lyon, France; with support from the World Health Organization secretariat: Dr J.
Fournier-Caruana, Dr I. Knezevic, Dr D.J. Wood, Dr T.Q. Zhou (Department of Immunization,
Vaccines and Biologicals), World Health Organization, Geneva, Switzerland.
The following provided comments on the second draft during a WHO consultation process from
26 July to 15 September 2011:
Page 45
I.S. Budiharto, Bio Farma, Bandung, Indonesia; W.A.M. Bakker and M van Oijen, National
Institute of Public Health and the Environment, Bilthoven, Netherlands; Ms V. Pithon and S.
Morgeaux, Agence Française de Sécurité Sanitaire des Produits de Santé, France; Y. Tano, Japan
Poliomyelitis Research Institute, Tokyo, Japan; Dr L. Fiore and Dr A.L. Salvati, Istituto
Superiore di Sanità, Rome, Italy; Dr G. Waeterloos, Scientific Institute of Public Health,
Brussels, Belgium; M.J. Uribe Serralde, A.M. Vionet and Dr J.B. Gonzalez, Birmex, Mexico; Dr
J. Martin, National Institute for Biological Standards and Control, Potters Bar, United Kingdom;
Dr C. Milne, European Directorate for the Quality of Medicines and HealthCare, Council of
Europe, France; Ms E. Coppens, Sanofi Pasteur, France; H. Wang, Beijing Tiantan Biological
Products Co. Ltd., Beijing, Peoples Republic of China; Dr L.Y. Yuan, National Institute for the
Control of Pharmaceutical and Biological Products, Beijing, P.R.China; and Dr L.A. Bigger and
O. Morin who coordinated and compiled comments from the Vaccines Committee of the
International Federation of Pharmaceutical Manufacturers and Associations, Geneva,
Switzerland.
The third draft was prepared by Dr M. Ferguson, Horning, United Kingdom and Dr T.Q. Zhou,
Immunization, Vaccines and Biologicals, World Health Organization, Geneva, Switzerland with
input from Dr J. Martin, Dr G. Cooper and Dr G. Dunn, National Institute for Biological
Standards and Control, Potters Bar, United Kingdom; and the drafting group members: Dr P.
Minor, National Institute for Biological Standards and Control, Potters Bar, United Kingdom; Dr
K. Chumakov, Center for Biologics Evaluation and Research, Food and Drug Administration,
Bethesda, MD, USA; Dr M. Baca-Estrada, Health Canada, Ottawa, Canada; Ms V. Pithon,
Agence Française de Sécurité Sanitaire des Produits de Santé, Lyon, France; and Dr J. Fournier-
Caruana, Immunization, Vaccines and Biologicals, and Dr R. Sutter, Polio Eradication Initiative,
World Health Organization, Switzerland.
The fouth draft was prepared by Dr M. Ferguson, Horning, United Kingdom and Dr T.Q. Zhou,
Immunization, Vaccines and Biologicals, World Health Organization, Geneva, Switzerland
following comments received during a WHO public consultation from November 2011 to
February 2012 from Dr A.Azhari, Bio Farma, Bandung, Indonesia; Dr L.A. Bigger who
coordinated and compiled comments from the Vaccines Committee of the International
Federation of Pharmaceutical Manufacturers and Associations, Geneva, Switzerland; Dr L. Fiore,
Istituto Superiore di Sanità, Rome, Italy; Dr J. Fournier-Caruana, Immunization, Vaccines and
Biologicals, World Health Organization , Geneva, Switzerland; Dr E. Leal, FIOCRUZ, Rio de
Janeiro, on behalf of the National Institute of Quality Control in Health, Brazil; Dr A. Lopez,
Laboratorios de Biológicos y Reactivos de México, Mexico; Dr S. Morgeaux, Agence Française
de Sécurité Sanitaire des Produits de Santé, Lyon, France; Dr S.-R. Pakzad, Food and Drug
Control Laboratory, Tehran, Islamic Republic of Iran; Ms V. Pithon, Agence Française de
Sécurité Sanitaire des Produits de Santé, Lyon, France; Dr A.L. Salvati, Istituto Superiore di
Sanità, Rome, Italy; Dr Y. Tano, Japan Poliomyelitis Research Institute, Tokyo, Japan; and Dr G.
Waeterloos, Scientific Institute of Public Health, Brussels, Belgium.
The fifth draft was prepared by Dr M. Ferguson, Horning, United Kingdom and Dr T.Q. Zhou,
Immunization, Vaccines and Biologicals, World Health Organization, Geneva, Switzerland,
taking into consideration comments provided on the fourth draft during a WHO consultation held
on 2729 March 2012 and attended by Dr S. Abe, Japan Poliomyelitis Research Institute, Tokyo,
Japan; Dr M. Baca-Estrada, Health Canada, Ottawa, Canada; Dr W.A.M. Bakker, National
Institute for Public Health and the Environment, Bilthoven, Netherlands; Dr J. Fournier-Caruana,
Immunization, Vaccines, and Biologicals, World Health Organization, Geneva, Switzerland; Mr
B.S. Chauhan, Bharat Biotech International Limited, Hyderabad, India; Dr K. Chumakov, Center
for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA; Dr
Page 46
E. Coppens, Sanofi Pasteur, France; Dr M. Duchêne, GlaxoSmithKline Biologicals, Wavre,
Belgium; Ms G. Dunn, National Institute for Biological Standards and Control, Potters Bar,
United Kingdom; Dr D. Felnerova, Crucell, Berne, Switzerland; Dr M. Ferguson, Horning,
United Kingdom; Dr L. Fiore, Istituto Superiore di Sanita, Roma, Italy; Mr J.B. González,
Laboratorios de Biológicos y Reactivos de México, Mexico; Dr M.A. Gonzalez, Federal
Commission for the Protection from Sanitary Risks, Ministry of Health, Mexico; Professor V.
Grachev, Russian Academy of Medical Sciences, Moscow, Russian Federation; Ms H. Wang ,
Tiantan Biological Products Co. Ltd , Beijing, China; Mrs T. Jivapaisarnpong, Department of
Medical Sciences, Ministry of Public Health, Bangkok, Thailand; Dr I. Knezevic, Immunization,
Vaccines, and Biologicals, World Health Organization, Geneva, Switzerland; Dr H. Okayasu,
Research, Policy and Product Development, World Health Organization, Geneva, Switzerland;
Dr D. Kusmiaty, National Quality Control Laboratory of Drug and Food, Ministry of Health,
Jakarta, Indonesia; Dr K. Katayama, National Institute of Infectious Diseases, Tokyo, Japan; Dr
C.G. Li, National Institutes for Food and Drug Control, Beijing, China; Dr J. Martin, National
Institute for Biological Standards and Control, South Mimms, United Kingdom; Dr C. Milne,
European Directorate for the Quality of Medicines and HealthCare, Strasbourg, France; Dr
R.Modi, Cadila Pharmaceuticals Ltd., Ahmedabad, India; Ms E. Niogret, Sanofi Pasteur, Marcy
L'Etoile, France; Dr L.V. Phung, National Institute for Control of Vaccine and Biologicals,
Hanoi, Viet Nam; Ms V. Pithon, Agence Française de Sécurité Sanitaire des Produits de Santé,
Lyon, France; Dr A. Sinyugina, Federal State Unitary Enterprise of Chumakov Institute of
Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, Moscow,
Russian Federation; Dr R. Sutter, Research, Policy and Product Development, World Health
Organization, Geneva, Switzerland; Mr D. Ugiyadi, Bio Farma, Bandung, Indonesia; Dr G.
Waeterloos, Scientific Institute of Public Health, Brussels, Belgium; Dr S. Yamazaki, Japan
Poliomyelitis Research Institute, Tokyo, Japan; Mr L. Yi, Institute of Medical Biology, Kunming,
China; Dr T.Q. Zhou, Immunization, Vaccines and Biologicals, World Health Organization,
Geneva, Switzerland.
The final draft document was prepared by Dr M. Ferguson and Dr T.Q. Zhou following
comments received on the fifth draft from: Dr K. Chumakov, Center for Biologics Evaluation
and Research, Food and Drug Administration, Bethesda, MD, USA; Dr J. Fournier-Caruana,
Immunization, Vaccines and Biologicals, World Health Organization , Geneva, Switzerland; Dr
E. Coppens, Sanofi Pasteur, Marcy L’Etoile, France; Dr C. Dubeaux, GlaxoSmithKline
Biologicals, Wavre, Belgium; Dr D. Ugiyadi, Bio Farma, Bandung, Indonesia; Dr M.A.
Gonzalez, Federal Commission for the Protection from Sanitary Risks, Ministry of Health,
Mexico; Dr C.G. Li, National Institutes for Food and Drug Control, Beijing, China; Dr P. Minor,
Dr A. Heath, Ms G. Dunn and Ms G. Cooper, National Institute for Biological Standards and
Control, Potters Bar, United Kingdom; Mr. B.S. Chauhan, Bharat Biotech International Ltd.,
Hyderabad, India; Dr. S. Yamazaki, Japan Poliomyelitis Research Institute, Tokyo, Japan; Dr V.
Pithon, Agence Nationale de Sécurité du Médicament et des Produits de Santé, Lyon, France; Dr.
E. Vitkova, European Directorate for the Quality of Medicines and HealthCare, Strasbourg,
France.
Further changes were made to document WHO/BS/2012.2185 by the Expert Committee on
Biological Standardization, resulting in the present document.
The following provided responses to a WHO survey on OPV seeds and quality control
information conducted during 20112012:
S.V. Shankarwar, Haffkine Bio Pharmaceutical Corporation Ltd., Mumbai, India; Y. Tano, Japan
Poliomyelitis Research Institute, Tokyo, Japan; A. Vidmanić, Institute of Virology, Belgrade,
Page 47
Serbia; R.C. Rosales and J.B. Gonzalez, Laboratorios de Biologicos y Reactivos De Mexico,
Birmex, Mexico; A. Azhari, Bio Farma, Bandung, Indonesia; W.A.M. Bakker and M. van Oijen,
National Institute of Public Health and the Environment, Bilthoven, Netherlands; M. Li, China
National Biotec Group, China; V. Grachev, A. Sinyugina and A. Malkin, Federal State Unitary
Enterprise on Production of Bacterial and Viral Preparations, Chumakov Institute of
Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, Moscow,
Russian Federation; T. Pasquali, Novartis Vaccines and Diagnostics Srl., Siena, Italy; E.
Coppens, Sanofi Pasteur, France; Q.H. Li, Kunming Institute of Medical Biology, Peking Union
Medical College, Kunming, China; N.D. Hien, Center for Research and Production of Vaccines
and Biologicals, Hanoi, Viet Nam; P. Amerlynck, GlaxoSmithKline Biologicals, Wavre,
Belgium; A. Mohammadi and A. Zand, Razi Vaccine and Serum Research Institute, Karaj,
Islamic Republic of Iran.
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Journal of Biological Standardization, 1973, 1:115118.
23. Stones PB et al. Preparation and properties of a derivative of Sabin’s type 3 poliovirus
strain Leon 12a,b. 10th Symposium of the European Association against Poliomyelitis,
Warsaw, 1964, pp 390397.
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Page 52
Appendix 1
Overview of virus seeds used in OPV production
The history of the poliovirus strains used in the production of OPV is well documented (1, 2, 3).
This appendix gives an overview of virus seeds used in current OPV production.
The flow diagrams in Figures 1 and 2 show the history of seed virus and reference materials used
in manufacture of OPV from Sabin 1 and Sabin 2 (Figure 1) and Sabin 3 (Figure 2) strains.
Concentric circles indicate progressive virus passages made to prepare master seed stocks,
working seed stocks and production lots of vaccine. Where relevant, sub-master seed stocks are
identified in the footnotes. Different seed viruses are identified as SO (Sabin Original), SOM
(Merck stock of SO), SOB (Behringwerke stock of SO), Pfizer (otherwise known as re-derived
SO, or RSO), SOJ (Japanese stock of SO) and SOR (Russian stock of SO).
These figures provide only a historical overview of the use of different seeds derived from the
Sabin vaccine strain in OPV production (as of June 2012). They do not indicate any WHO
"qualification" or "approval" of the strains or vaccines in the context of this document.
Page 53
Figure 1. Types 1 and 2 OPV
Page 54
Figure 2. Type 3 OPV
Page 55
Names of manufacturers shown on Figures 1 and 2 are as follows:
Belgium GlaxoSmithKline Biologicals
China (1) Institute of Medical Biology, Kunming
China (2) China National Biotec Group, Beijing Tiantan Biological Products Company
France Sanofi Aventis
Indonesia PT Bio Farma
Iran Razi Vaccine and Serum Research Institute
Italy Novartis Vaccines
Japan Japan Poliomyelitis Research Institute
Mexico Biologics and Reagents Laboratories of Mexico
Russia Federal State Unitary Enterprise of Chumakov Institute of Poliomyelitis and
Viral Encephalitides
Serbia Torlak Institute of Virology, Vaccines and Serum
Viet Nam Center for Research and Production of Vaccines and Biologicals
Notes:
1. Working seeds produced by different manufacturers before 1976.
2. WHO master seed stock.
3. WHO neurovirulence reference preparation.
4. Type 1 seed stock prepared at JPRI by four passages of SOM, including three terminal
dilution passages (passage level SO+5). Type 2 seed stock prepared at JPRI by one passage
of SOM (SO+2).
5. Seed stock prepared at JPRI by one passage of SOB (SO+2).
6. Novartis performed an additional passage to prepare sub-master seed stock from which a
working seed was produced.
7. Six plaques were selected, pooled together, and grown to produce seed stock in Russia.
8. Pfizer (RSO): re-derived Sabin Original, produced by RNA plaque purification, passage.
9. Zhong-3: plaque purification, passage.
10. Produced by JPRI in 1969 from SO stock by one passage (SO+1).
11. Prepared from SOJ by passages in AGMK cells (SOJ+9), including two plaque purifications
and three terminal passages (SO+10).
12. Prepared from SOJ by passages in AGMK cells (SOJ+6), including two plaque purifications
(SO+7).
References
1. Cockburn WC. The work of the WHO Consultative Group on Poliomyelitis Vaccines.
Bulletin of the World Health Organization, 1988, 66:143154.
2. Sabin AB, Boulger L. History of Sabin attenuated poliovirus oral live vaccine strains.
Journal of Biological Standardization, 1973, 1:115118.
3. Stones PB et al. Preparation and properties of a derivative of Sabin’s type 3 poliovirus
strain Leon 12a,b. 10th Symposium of the European Association against Poliomyelitis,
Warsaw, 1964, pp 390397.
Page 56
Appendix 2
In vivo tests for neurovirulence and considerations for the choice of assay
Live attenuated vaccines were developed by Sabin in large part by use of nonhuman primates,
particularly old world monkeys for measuring the level of residual neurovirulence. In the 1980s,
tests of vaccine bulks and seeds were standardized as a single dose of test material given by
intraspinal inoculation tested concurrently with a homologous reference. Vaccines derived from
the Sabin strains that pass the monkey neurovirulence test (MNVT) have been shown to have an
acceptable safety profile. However, in its current form, the MNVT is regarded as a test of
consistency and it is not known whether vaccines that fail the test are virulent in human
recipients. Tests designed to replace the MNVT should be able to detect the same changes from
batch to batch with similar sensitivity. A neurovirulence test in transgenic mice (TgmNVT)
expressing the human poliovirus receptor (TgPVR21 mice) has been developed as an alternative
to the MNVT for all three poliovirus serotypes.
Summaries of the MNVT and TgmNVT are given below, along with the implementation process
for the TgmNVT.
1. Summary of the monkey neurovirulence test (MNVT)
1.1 Key features
A detailed set of standard operating procedures for the “Neurovirulence test of types 1, 2 or 3
live attenuated poliomyelitis vaccines (oral) in monkey” is available from WHO.1 Between 5.5
and 6.5 log10 CCID50 of monovalent virus is delivered in a single dose by intraspinal inoculation
into the lumbar cord. A back titration of the inoculum should be carried out after the inoculation
step is completed. Residual paralysis, if any, is noted over the following 1722 days. The
animals are sacrificed at the end of the test or earlier on humane grounds and prepared for
histological examination of the central nervous system. Regions are scored for damage on a scale
from 1 to 4, and a mean lesion score is calculated for each monkey and then for all the monkeys
in the test. The clinical signs do not form part of the assessment or of the pass/fail criteria. The
homologous WHO/SO+2 reference is tested in parallel. For a new laboratory, the
implementation process should be agreed with the NRA.
1.2 Number of animals
The number of monkeys has been chosen on statistical grounds, considering the variability of the
test, such that a satisfactory vaccine will only twice give the lesion score of a reference
preparation in 1% of tests and therefore be incorrectly scored as a fail. Valid animals must show
some sign of histological damage as evidence of correct placement of active virus. The number
of valid monkeys required per virus preparation is 11 for types 1 and 2 and 18 for type 3.
Because a reference must be tested at the same time, the total number of monkeys is at least 22
for types 1 and 2 and 36 for type 3.
1.3 Sections examined
1 Contact the Coordinator, Quality, Safety and Standards, World Health Organization, 1211, Geneva 27, Switzerland
(http://www.who.int/biologicals/vaccines/en/).
Page 57
Sections are examined from defined regions of the spinal cord and brain and scored
histologically for virus activity on a scale of 1 (cellular infiltration only) to 4 (massive neuronal
damage). At least 29 sections are examined per monkey, as specified in the WHO standard
operating procedures for the MNVT. The readings are used to generate the mean lesion score for
the animal, and the mean lesions scores for all animals are then used to generate the mean lesion
score for the test as a whole.
1.4 Pass/fail criteria
The pass/fail criteria are based on the variation in the test from run to run, established from the
scores obtained with the reference preparation and specific to each laboratory and operator. The
within-test variance is used to calculate the statistical constants C1, C2 and C3. If the mean lesion
score of the test vaccine is greater than that of the concurrently tested reference by more than C1,
the vaccine is not acceptable. If the test vaccine gives a higher score than the reference but the
difference in scores lies between C1 and C2, the vaccine may be retested and the results pooled; if
the difference for the pooled test results is greater than C3, the vaccine fails.
The values for C1, C2 and C3 are initially established on the basis of the data accumulated after
four qualifying tests. These values should then be updated after every test until nine tests have
been performed. After that, the C values are based on the last 10 tests performed. The C values
must be established for each testing laboratory.
2. Summary of the transgenic mouse neurovirulence test (TgmNVT)
2.1 Key features
The detailed operating procedures for the TgmNVT, "WHO neurovirulence test of type 1, 2 or 3
live attenuated poliomyelitis vaccines (oral) in transgenic mice susceptible to poliovirus", are
available from WHO.1 The test for neurovirulence of polio vaccines in transgenic mice involves
the intraspinal inoculation of a defined strain of transgenic mice carrying the human receptor for
poliovirus with small volumes of the test vaccine. Two virus concentrations are used and the
read-out of the test is based on the clinical dose response. A reference preparation is tested at the
same time and a clearly defined process has been established for implementation of the test in a
new laboratory.
2.2 Strain of transgenic mouse
Different transgenic mouse lines differ in their sensitivity to polio infection depending on the
particular transgenic construct and the genetic background, and only strains from a source
approved by WHO should be used. Currently the only approved transgenic mouse strain is
TgPVR21, developed in Japan and sourced from the developers or from an approved
subcontractor.
2.3 Titration of virus
Two doses of virus are inoculated in a volume of five microlitres: for type 1, 1.75 and 2.75
CCID50; for type 2, 5.0 and 6.0 CCID50; and for type 3, 3.5 and 4.5 CCID50. The inocula must be
prepared and titrated accurately to ensure that these doses are given; the precision of the
1 Contact the Coordinator, Quality, Safety and Standards, World Health Organization, 1211, Geneva 27, Switzerland
(http://www.who.int/biologicals/vaccines/en/).
Page 58
determinations should be better than +/- 0.3 log10. A back titration of the inoculum should be
performed after the inoculation step is completed.
2.4 Inoculation and observation of animals
Animals procured at age 56 weeks are randomized to cages and allowed to recover for at least
seven days. They are then appropriately anaesthetized and inoculated with five microlitres of
diluted test virus between the last thoracic and first lumbar vertebrae. Animals are observed for
clinical signs once a day for the next 14 days and ultimately scored either as normal (slight
weakness or no signs) throughout or paralysed (paresis on two consecutive days or paralysis on a
single day). The lower and higher doses of the reference should give more than 5% and less than
95% of animals paralysed, respectively, for the test to be valid. A test requires 128 mice for one
vaccine plus the reference tested concurrently, or 192 for two vaccines and the reference. The
reference is the same as that used in the monkey test; the use of other references may be
acceptable but should be validated.
The vaccine passes if it is not significantly more virulent than the reference defined in terms of
the log odds ratio and statistical constants L1 and L2 which are based on the reproducibility of
the test and define the pass/fail criteria and the grey zone in which a retest is required. The
acceptance and rejection limits, L1 and L2, were selected so that a test vaccine which is
equivalent to the reference will have a 0.95 probability of passing and a 0.01 probability of
failing, respectively. The constants are regularly updated. Statistical evaluation of test validity
includes linearity and dose and gender effects.
3. Implementation process of the TgmNVT
If a manufacturer wishes to use the transgenic mouse test, relevant validation data should be
available for their specific product to demonstrate the test’s applicability. This may include
reference to the extensive collaborative studies by which the test was originally developed. A
clear stepwise process for implementing the TgmNVT has been established; it involves training
in the inoculation technique through injection of Indian ink, tests with vaccines, and testing of a
blinded evaluation panel containing vaccines that pass, fail or marginally fail the test.
Competence in clinical scoring is acquired through a standardized training procedure which
involves parallel scoring with an experienced scorer and criteria for declaring a trainee
competent.
Testing should be performed according to procedures specified in the WHO standard operating
procedures for the TgmNVT, using appropriate WHO reference materials unless modified
procedures have been validated and shown to be suitable. The test chosen should be used to test
virus seeds and bulks, as described in sections A.3.2.4.2 and A.4.4.7.2 respectively.
4. Considerations for the choice of assay
The following specific issues suggest that care should be taken in the selection of the in vivo
tests to be performed for neurovirulence and that the selection should be justified. The report of
the WHO Working Group Meeting to Discuss the Revision of the WHO Recommendations for
OPV: TRS Nos. 904 and 910 provides more detailed discussions (1).
4.1 Types 1 and 2 Sabin vaccine viruses
The relative sensitivity of the transgenic mouse and monkey tests performed according to WHO
procedures with respect to the presence of mutations in the 5’untranslated region (UTR) in types
Page 59
1 and 2 appears to be comparable but significantly lower than that in type 3 (2, 3). It is unknown
whether these two models are equally sensitive to other potential neurovirulent mutations. Most
manufacturers use essentially identical seeds of types 1 and 2, in contrast to the situation with
type 3.
4.2 Type 3 Sabin vaccine virus
4.2.1 Molecular biology
Studies of the molecular biology of the Sabin polio vaccine virus strains have suggested that few
mutations are involved in attenuation and that, for the type 3 strain, there may be only two: one
base change in the 5’noncoding region of the genome at base 472 and one coding change at base
2034 that introduces an amino acid change in the virus protein VP3. A third mutation at position
2493 has been described (4). Growth of Sabin 3 virus in cell culture or in vaccine recipients
results in rapid accumulation of U instead of C at nucleotide 2493 (changing Thr to Ile at amino
acid 6 of capsid protein VP1), and all Sabin 3 OPV batches contain variable amounts of these
mutants. This mutation does not affect neurovirulence as determined in the monkey test but there
is evidence that it influences the results obtained in the transgenic mouse test, as described in the
WHO operating procedures (5). Variations in the virulence of vaccine batches measured in
monkeys correlate well with variations in the base in the 5’ noncoding region as measured by
MAPREC. Changes in the amino acid in VP3, or changes at other positions that suppress its
effect, are not thought to be generated in the course of well controlled production runs, although
this is possible in principle.
4.2.2 Current type 3 seed viruses
Seed viruses currently used for global vaccine production contain variable proportions of the
bases found at position 2493 (C or U):
The original WHO reference material (passage level SO+2) for neurovirulence
testing contained about an equal mixture of both forms (2493 C or U).
Batches prepared from RSO, the seeds most commonly used in production in Europe,
typically contain about 5% or less of 2493-U (mutant).
Seed viruses used in production by some manufacturers (a plaque purified from SO)
contain 100% of mutant form (2493-U) (6).
All OPVs currently in use are believed to have an acceptable safety profile.
5. Experience from the use of MNVT and TgmNVT with type 3 seeds and vaccines
There is evidence that the transgenic mouse test, as described in the WHO operating procedures
for the TgmNVT, is sensitive to the presence of 2493-U, whereas the monkey test is not sensitive
to this mutation. Thus, batches produced from RSO seed will pass both the monkey and
transgenic mouse tests, whereas batches produced from the alternative seeds that contain 100%
2493-U will pass the monkey test but may fail the transgenic mouse test, although still having an
acceptable safety profile in clinical use.
WHO’s current standard operating procedures for the TgmNVT specify the doses and the WHO
reference material to be used and include the proportion of mice affected at the two doses of
virus given for the test to be valid. The WHO reference material for TgmNVT is the same as that
used in the monkey test and has approximately 50% 2493-C, and it was validated primarily
Page 60
against vaccines made from SO or RSO seeds. However, if used to test vaccines derived from
2493-U containing seed, it may fail them even if they contain little 472-C and would pass
MNVT. The TgmNVT could be adapted for testing 2493-U containing bulks – e.g. by changing
the reference material, the doses and/or the validity criteria. Manufacturers may wish to do this
to make it applicable to their product. Any modified test should be validated and approved by the
NRA.
References
1. WHO Working Group Meeting to Discuss the Revision of the WHO Recommendations for
OPV: TRS No. 904 and 910. Geneva, Switzerland 2022 July 2010. Geneva, World
Health Organization, 2010
(http://www.who.int/biologicals/vaccines/OPV_Meeting_report_Final_Clean_13May2011.pdf,
accessed 16 January 2013).
2. Gennady V et al. Genetic stability of Sabin 1 strain of poliovirus: implications for quality
control of oral poliovirus vaccine. Virology, 1998, 245:183–187.
3. Taffs RE et al. Genetic stability and mutant selection in Sabin 2 strain of oral poliovirus
vaccine grown under different cell culture conditions. Virology, 1995, 209:6673.
4. Tatem JM et al. A mutation present in the amino terminus of Sabin 3 poliovirus vp1
protein is attenuating. Journal of Virology, 1992, 66:3194–3197.
5. Chumakov KM et al. RNA sequence variants in live poliovirus vaccine and their relation
to neurovirulence. Journal of Virology, 1992, 66:966970.
6. Rezapkin GV et al. Reevaluation of nucleotide sequences of wild-type and attenuated
polioviruses of type 3. Virus Research, 1999, 65(2):111119.
Page 61
Appendix 3
Preparation of live attenuated poliomyelitis vaccine (oral) using cell banks Example of a flowsheet of tests in cell cultures
* Control cells: 5% of the total or 500 ml of cell suspension, or 100 million cells.
HAEM = test for haemadsorbing viruses.
CL = cell line used for production, but not the same batch of cells used for production of
the virus.
SC = when a human diploid cell line is used for production, a simian kidney cell line
should be used as the second indicator cell line. When a simian kidney cell line is used for
production, a human diploid cell line should be used as the second indicator cell line (1).
HC = human cells.
Note: This example includes all tests, whether obligatory or not. Since the requirements applicable in a particular place
are those authorized by the NRA, this flowsheet should not be considered as an integral part of the requirements and has
been included solely for guidance. Manufacturing establishments should prepare their own flowsheet in order to clarify
the procedures used.
Page 62
Reference
1. Recommendations for the evaluation of animal cell cultures as substrates for the
manufacture of biological medicinal products and for the characterization of cell banks.
Geneva, World Health Organization, 2010
(http://www.who.int/entity/biologicals/Cell_Substrates_clean_version_18_April.pdf,
accessed 16 January 2013).
Page 63
Appendix 4
Cell-culture techniques for the determination of the virus content of
live attenuated poliomyelitis vaccine (oral)
This appendix describes a method for the determination of the virus content of live attenuated
OPV in cell cultures. It is an example that is provided for guidance only.
The preparation to be assayed and the reference preparation are diluted in an appropriate medium.
It is convenient to make tenfold dilution steps of the virus suspensions initially, but for dilutions
that are to be inoculated into Hep-2 (Cincinnati) cell cultures the dilutions should be prepared in
1.0 log10 or smaller steps. A preliminary assay may be required to ensure that, in the test, the
dilution range selected encompasses at least three dilutions that will infect between 0% and
100% of the cultures inoculated.
Titrate the vaccine for infectious virus using no fewer than three separate containers of vaccine
following the method described below. Titrate one container of an appropriate virus reference
preparation in triplicate to validate each assay. The virus titre of the reference preparation is
monitored using a control chart, and a titre is established on a historical basis by each laboratory.
If the vaccine contains more than one poliovirus type, titration of the individual serotypes is
undertaken separately using mixtures of appropriate type-specific antiserum (or preferably a
monoclonal antibody) to neutralize each of the other types present.
For titration of individual serotypes, inoculate a suitable number of wells (ideally 810) in a flat-
bottomed microtitre plate with equal volumes of the selected dilutions of virus and the
appropriate antisera mixture. Total virus content is determined, without any prior incubation, by
directly diluting the vaccine in the assay medium. The assay is then incubated for 13 hours at
3436 o C,
followed by the addition of an appropriate volume of a suitable cell. The plates are
further incubated at 3436 o C and examined between day 5 and day 9 for the presence of viral
cytopathic effect.
The cytopathic effect can be observed by direct reading or after an appropriate staining (vital or
fixed staining). The individual virus concentration for each polio serotype and reference
preparation is then calculated using an appropriate method.
The assay is considered valid if:
the estimated virus concentration for the reference preparation is ± 0.5 log10
CCID50 of the established value for this preparation;
the confidence interval (P = 0.95) of the estimated virus concentration of the
three replicates of the reference preparation is not greater than ± 0.3 log10
CCID50.
The assay is repeated and results are averaged if:
the confidence interval (P = 0.95) of the combined virus concentration of the
vaccine is greater than ± 0.3 log10 CCID50.
Page 64
Appendix 5
Model summary protocol for manufacturing and control of live attenuated
poliomyelitis vaccines (oral)
The following protocol is intended for guidance, and indicates the information that should be
provided as a minimum by the manufacturer to the NRA.
Information and tests may be added or deleted as required by the NRA, if applicable.
It is thus possible that a protocol for a specific product may differ in detail from the model
provided. The essential point is that all relevant details demonstrating compliance with the
licence and with the relevant WHO recommendations on a particular product should be given in
the protocol submitted.
The section concerning the final lot must be accompanied by a sample of the label and a copy of
the leaflet that accompanies the vaccine container. If the protocol is being submitted in support
of a request to permit importation, it should also be accompanied by a lot release certificate from
the NRA of the country in which the vaccine was produced/released, stating that the product
meets the national requirements as well as the recommendations of Part A of this document
published by WHO.
Summary information on finished product (final vaccine lot)
International name: _______________________________________
Trade name: _______________________________________
Product licence (marketing authorization)
number:
_______________________________________
Country: _______________________________________
Name and address of manufacturer: _______________________________________
Name and address of licence holder, if different: _______________________________________
Virus strain: _______________________________________
Origin and short history: _______________________________________
Finished product (final lot):
Batch number: _______________________________________
Final bulk: _______________________________________
Type of container: _______________________________________
Number of doses per container: _______________________________________
Number of filled containers in this final lot: _______________________________________
Bulk numbers of monovalent bulk Type 1 Type 2 Type 3
suspensions blended in
monovalent/bivalent/trivalent vaccine:
Page 65
Site of manufacture of each monovalent bulk: _______________________________________
Date of manufacture of each monovalent bulk: _______________________________________
Date of manufacture of final bulk (blending): _______________________________________
Date of manufacture (filling) of finished
product:
_______________________________________
Date on which last determination of virus titre
was started, or date of start of period of validity:
_______________________________________
Shelf-life approved (months): _______________________________________
Expiry date:
Storage conditions: _______________________________________
Volume of human dose (in drops
and/or ml):
_______________________________________
Virus titre per single human dose: _______________________________________
Type 1: _______________________________________
Type 2: _______________________________________
Type 3: _______________________________________
Nature and concentration of
stabilizer:
_______________________________________
Nature of any antibiotics present in
vaccine and amount per human dose:
_______________________________________
Release date: _______________________________________
Starting materials
The information requested below is to be presented on each submission. Full details on master and
working seed lots should be provided upon first submission only and whenever a change has been
introduced.
The following sections are intended for recording the results of the tests performed during the production
of the vaccine, so that the complete document will provide evidence of consistency of production. If any
test has to be repeated, this must be indicated. Any abnormal result must be recorded on a separate sheet.
If any cell lot or virus harvest intended for production is rejected during the control testing, this should
also be recorded either in the following sections or on a separate sheet.
Control of source materials (section A.3)
Cell banks (every submission)
Information on cell banking system:
Name and identification of substrate: Origin and short history:
_______________________________________
Authority that approved the cell bank:
_______________________________________
Master cell bank (MCB) and working cell _______________________________________
Page 66
bank (WCB) lot numbers and date of
preparation:
Date the MCB and WCB were
established:
_______________________________________
Date of approval by national regulatory
authority:
_______________________________________
Total number of ampoules stored:
_______________________________________
Passage level (or number of population
doublings) of cell bank: _______________________________________
Maximum passage approved: _______________________________________
Storage conditions: _______________________________________
Method of preparation of cell bank in
terms of number of freezes and efforts
made to ensure that a homogeneous
population is dispersed into the ampoules:
_______________________________________
Tests on MCB and WCB, first submission only (section A.3.1.2)
Percentage of total cell-bank ampoules
tested:
_______________________________________
Identification of cell substrate:
_______________________________________
Method:
_______________________________________
Specification:
_______________________________________
Date of test:
_______________________________________
Result:
_______________________________________
Growth characteristics:
_______________________________________
Morphological characteristics:
_______________________________________
Immunological marker:
_______________________________________
Cytogenetic data:
_______________________________________
Biochemical data:
_______________________________________
Results of other identity tests
: _______________________________________
Tests for adventitious agents
_______________________________________
Method used:
_______________________________________
Number of vials tested:
_______________________________________
Volume of inoculum per vial:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test: _______________________________________
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Result:
_______________________________________
Tests for bacteria, fungi and
mycoplasma
Tests for bacteria and fungi
Method used:
_______________________________________
Number of vials tested:
_______________________________________
Volume of inoculum per vial:
_______________________________________
Volume of medium per vial:
_______________________________________
Observation period (specification):
_______________________________________
Incubation Media used Inoculum Date of start of
test
Date of end of
test
Results
20–25 °C
__________ __________ ___________ ___________ __________
30–36 °C
__________ __________ ___________ ___________ __________
Negative
control: __________ __________ ___________ ___________ __________
Test for mycoplasma
Method used:
_______________________________________
Volume tested:
_______________________________________
Media used:
_______________________________________
Temperature of incubation:
_______________________________________
Observation period (specification):
_______________________________________
Positive controls (list of species used and
results):
Date of start of test Date of end of test Results
Subcultures at day 3:
_______________ _______________ _______________
Subcultures at day 7:
_______________ _______________ _______________
Subcultures at day 14:
_______________ _______________ _______________
Subcultures at day 21:
_______________ _______________ _______________
Indicator cell-culture method (if
applicable)
Cell substrate used:
_______________________________________
Inoculum:
_______________________________________
Date of test:
_______________________________________
Passage number:
_______________________________________
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Negative control:
_______________________________________
Positive controls:
_______________________________________
Date of staining:
_______________________________________
Results:
_______________________________________
Results of tests for tumorigenicity (if
applicable) :
_______________________________________
Virus seed A.3.2 (Every submission)
Vaccine virus strain(s) and serotype(s):
_______________________________________
Substrates used for preparing seed lots:
_______________________________________
Origin and short history:
_______________________________________
Authority data approved virus strains:
_______________________________________
Date of approval: _______________________________________
Information and seed lot preparation, every submission (section A.3.2.1
Virus master seed (VMS), virus sub-master seed, and virus working seed (VWS)
Source of VMS: _______________________________________
VMS and VWS lot number: _______________________________________
Name and address of manufacturer: _______________________________________
VWS passage level from VMS: _______________________________________
Dates of inoculation: _______________________________________
Dates of harvest: _______________________________________
Numbers of containers: _______________________________________
Conditions of storage: _______________________________________
Dates of preparation: _______________________________________
Maximum passages levels authorized: _______________________________________
Tests on virus master seed (VMS), virus sub-master seed, and virus working seed (VWS), first
submission only
Test for adventitious agents
Date(s) of satisfactory test(s) for freedom
from adventitious agent:
_______________________________________
Volume of virus seed samples for
neutralization and testing: _______________________________________
Batch number of antisera used for
neutralization virus seed: _______________________________________
Method used:
_______________________________________
Date of start of test:
_______________________________________
Page 69
Date of end of test:
_______________________________________
Result:
_______________________________________
Identity test
_______________________________________
Method used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Result:
_______________________________________
Absence of SV40
Method used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Results:
_______________________________________
In vitro tests: MAPREC or rct/40 marker
test
MAPREC
Date of test:
_______________________________________
Type 1
Ratio of % of the sum of both mutations
480-A, 525-C of bulk sample to the
International Standard or
level of mutations:
_______________________________________
Result of test of consistency of production:
_______________________________________
Result of test of comparison with the
International Standard: _______________________________________
Type 2
Ratio of % 481-G of bulk sample to the
International Standard or
level of mutations:
_______________________________________
Result of test of consistency of production:
_______________________________________
Result of test of comparison with the
International Standard: _______________________________________
Type 3
Ratio of %472C of bulk sample to the
International Standard or
level of mutations:
_______________________________________
Result of test of consistency of production
_______________________________________
Result of test of comparison with the
International Standard _______________________________________
In vitro rct/40 marker test
Date of test:
_______________________________________
Page 70
Reduction of titre of bulk sample:
_______________________________________
Reduction of titre of negative reference:
________________________________________
Reduction of titre of positive reference:
________________________________________
Result:
_______________________________________
Result of test of consistency of production:
_______________________________________
In vivo tests for neurovirulence
Neurovirulence test in monkeys:
_______________________________________
Result of blood serum test in monkeys prior
to inoculation:
_______________________________________
Number and species of monkeys inoculated:
_______________________________________
Quantity (CCID50) inoculated in each test
monkey:
_______________________________________
Number of “valid” monkeys inoculated
with test sample: _______________________________________
Number of positive monkeys observed
inoculated with test sample or with
reference:
_______________________________________
Reference preparation:
_______________________________________
Number of "valid" monkeys inoculated with
reference:
_______________________________________
Number of positive monkeys observed:
_______________________________________
Mean Lesion Score of test sample: _______________________________________
Mean Lesion Score of reference:
(see also attached forms giving
details of histological observations
and assessment)
_______________________________________
C1 constant value: _______________________________________
Neurovirulence test in transgenic mice
Strain of mice inoculated:
_______________________________________
For each dose of the seed sample:
_______________________________________
Number of mice inoculated:
_______________________________________
Number of mice excluded from
evaluation:
_______________________________________
Number of mice paralysed: _______________________________________
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Results of validity tests for each dose of the
reference virus:
_______________________________________
Number of mice inoculated:
_______________________________________
Number of mice excluded from evaluation:
_______________________________________
Number of mice paralysed:
_______________________________________
Virus assay results for each dose inoculated
(residual inoculums): _______________________________________
Paralysis rates for test vaccine at each dose:
Paralysis rates for reference virus at each
dose: _______________________________________
Results:
_______________________________________
Log odds ratio:
_______________________________________
L1 and L2 values:
_______________________________________
Pass/fail decision:
_______________________________________
Freedom from bacteria, fungi and
mycoplasmas
Tests for bacteria and fungi
Method used:
_______________________________________
Number of vials tested:
_______________________________________
Volume of inoculum per vial:
_______________________________________
Volume of medium per vial:
_______________________________________
Observation period (specification)
_______________________________________
Incubatio
n
Media used Inoculum Date of start of test Date of end of test Results
20–25 °C
__________ __________ ___________ ___________ __________
30–36 °C
__________ __________ ___________ ___________ __________
Negative
control __________ __________ ___________ ___________ __________
Test for mycoplasma
Method used:
_______________________________________
Volume tested:
_______________________________________
Media used:
_______________________________________
Temperature of incubation:
_______________________________________
Observation period (specification) :
_______________________________________
Positive controls (list of species used and
Page 72
results) :
Date of start of test Date of end of test Results
Subcultures at day 3 _______________ _______________ _______________
Subcultures at day 7 _______________ _______________ _______________
Subcultures at day 14 _______________ _______________ _______________
Subcultures at day 21 _______________ _______________ _______________
Indicator cell-culture method (if
applicable)
Cell substrate used:
_______________________________________
Inoculum:
_______________________________________
Date of test:
_______________________________________
Passage number:
_______________________________________
Negative control:
_______________________________________
Positive controls:
_______________________________________
Date of staining:
_______________________________________
Results:
_______________________________________
Virus titration
Date of test:
_______________________________________
Reference batch number: _______________________________________ Date of test:
_______________________________________
Result:
_______________________________________
Genotype characterization
Method used:
_______________________________________
Date of test:
_______________________________________
Result
_______________________________________
Test for mycobacteria
Method used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Result:
_______________________________________
Control of vaccine production (section A.4.1)
Control of production cell cultures
Lot number of MCB: _______________________________________
Lot number of WCB: _______________________________________
Date of thawing of ampoule of WCB:
Page 73
Passage number of production cells: _______________________________________
Date of preparation of control cell
cultures: _______________________________________
Results of microscopic observation: _______________________________________
Tests on control cell cultures
Ratio of control to production cell
cultures:
_______________________________________
Incubation conditions:
Period of observation of cultures:
Dates observation started/ended:
_______________________________________
Ratio or proportion of cultures discarded
for nonspecific reasons:
_______________________________________
Results of observation:
_______________________________________
Date of supernatant fluid collected:
_______________________________________
Tests for haemadsorbing viruses
Quantity of cell tested:
_______________________________________
Method used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Results:
_______________________________________
Tests for adventitious agents on
supernatant culture fluids
_______________________________________
Method used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Result:
______________________________________
Identity test
Method used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Result:
_______________________________________
Control of single harvests (section A.4.3)
Volume harvested: _______________________________________
Page 74
Date of sampling:
_______________________________________
Identity test
Method used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Result:
_______________________________________
Virus titration
Date of test:
_______________________________________
Reference batch number:
_______________________________________
Date of test:
_______________________________________
Result:
_______________________________________
Tests of neutralized single harvests for
adventitious agents
_______________________________________
Method used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Result:
_______________________________________
Freedom from bacteria, fungi and
mycoplasmas
Tests for bacteria and fungi
Method used
_______________________________________
Number of vials tested:
_______________________________________
Volume of inoculum per vial:
Volume of medium per vial:
_______________________________________
Observation period (specification)
_______________________________________
Incubation Media
used
Inoculum Date of start of test Date of end of test Results
20–25 °C
_______ _______ ____________ ______________ ________
30–36 °C
_______ _______ ____________ ______________ ________
Negative
control
_______ _______ ____________ ______________ ________
Test for mycoplasma
Method used: _______________________________________
Page 75
Volume tested:
_______________________________________
Media used:
_______________________________________
Temperature of incubation:
_______________________________________
Observation period (specification) :
_______________________________________
Positive controls (list of species used and
results): _______________________________________
Date of start of test Date of end of test Results
Sub cultures at 3rd
day
_______________ _______________ _______________
Sub cultures at 7th
day
_______________ _______________ _______________
Sub cultures at
14th day
_______________ _______________ _______________
Sub cultures at
21th day
_______________ _______________ _______________
Indicator cell-culture method (if
applicable)
Cell substrate used:
_______________________________________
Inoculum:
_______________________________________
Date of test:
_______________________________________
Passage number:
_______________________________________
Negative control:
_______________________________________
Positive controls:
_______________________________________
Date of staining:
_______________________________________
Results:
_______________________________________
Test for mycobacteria
Method used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Result:
_______________________________________
Control of monovalent bulk (section A.4.4)
Date of filtration of bulk:
_______________________________________
Porosity of filters used:
_______________________________________
Date of sampling:
_______________________________________
Identity test
Page 76
Method used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Results:
_______________________________________
Lot number of reference reagents:
_______________________________________
Virus titration
Date of test:
_______________________________________
Reference batch number:
_______________________________________
Result:
_______________________________________
Tests for bacteria and fungi
Method used
_______________________________________
Number of vials tested:
_______________________________________
Volume of inoculum per vial:
_______________________________________
Volume of medium per vial:
_______________________________________
Observation period (specification):
_______________________________________
Incubation Media used Inoculum Date of
start of
test
Date of
end of
test
Results
20–25 °C
_______ _______ ______ ______ _______
30–36 °C
_______ _______ ______ ______ _______
Negative control: _______ _______ ______ ______ _______
Test for mycobacteria
Method used:
______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Result:
_______________________________________
Tests for consistency of virus
characteristics
In vitro rct/40 marker test
Date of test:
_______________________________________
Reference used:
_______________________________________
Page 77
Reduction or titre of negative reference:
_______________________________________
Reduction of titre of positive reference:
_______________________________________
Result:
_______________________________________
MAPREC
Date of test:
_______________________________________
Type 1
Ratio of % of the sum of both mutations
480-A, 525-C of bulk sample to the
International Standard or
level of mutations:
_______________________________________
Result of test of consistency of
production: _______________________________________
Result of test of comparison with the
International Standard: _______________________________________
Type 2
Ratio of % 481-G of bulk sample to the
International Standard or
level of mutations:
_______________________________________
Result of test of consistency of
production
Result of test of comparison with the
International Standard: _______________________________________
Type 3
Ratio of % 472C of bulk sample to the
International Standard or
level of mutations
_______________________________________
Result of test of consistency of
production: _______________________________________
Result of test of comparison with the
International Standard:
_______________________________________
Neurovirulence tests in monkeys
Result of blood serum test in monkeys
prior to inoculation:
_______________________________________
Date of inoculation of monovalent bulk:
_______________________________________
Number and species of monkeys
inoculated:
_______________________________________
Quantity (CCID50) inoculated in each
test monkey:
_______________________________________
Number of “valid” monkeys inoculated
with test sample: _______________________________________
Number of positive monkeys observed
inoculated with test sample or with
reference:
_______________________________________
Reference preparation:
_______________________________________
Number of "valid" monkeys inoculated _______________________________________
Page 78
with reference:
Number of positive monkeys observed:
Mean Lesion Score of test sample:
Mean Lesion Score of reference:
(see also attached forms giving
details of histological observations
and assessment)
_______________________________________
C1 constant value: _______________________________________
Neurovirulence test in transgenic mice
Strain of mice inoculated:
_______________________________________
For each dose of the bulk sample:
_______________________________________
Number of mice inoculated:
_______________________________________
Number of mice excluded from
evaluation:
_______________________________________
Number of mice paralysed:
_______________________________________
Results of validity tests for each dose of
the reference virus: _______________________________________
Number of mice inoculated:
_______________________________________
Number of mice excluded from
evaluation:
_______________________________________
Number of mice paralysed:
_______________________________________
Virus assay results for each dose
inoculated
(residual inoculums) :
_______________________________________
Paralysis rates for test vaccine at each
dose:
_______________________________________
Paralysis rates for reference virus at each
dose: _______________________________________
Results:
_______________________________________
Log odds ratio:
_______________________________________
L1 and L2 values:
_______________________________________
Pass/fail decision:
_______________________________________
Final bulk A.4.5
Preparation of
bulk (types as
appropriate):
Type 1 Type 2 Type 3
Monovalent bulks
in blend:
____________ ______________ ___________________
Volume in blend:
____________ ______________ ___________________
Nature and ____________ ______________ ___________________
Page 79
volume of
stabilizer:
Nature and
volume of diluent:
____________ ______________ ___________________
Total volume of blend: _______________________________________
Tests for bacteria and fungi
Method used:
_______________________________________
Number of vials tested:
_______________________________________
Volume of inoculum per vial:
_______________________________________
Volume of medium per vial:
_______________________________________
Observation period (specification):
_______________________________________
Incubatio
n
Media used Inoculum Date of start of test Date of end of test Results
20–25 °C
__________ __________ ___________ ___________ __________
30–36 °C
__________ __________ ___________ ___________ __________
Negative
control __________ __________ ___________ ___________ __________
Filling and containers (section A.5)
Total volume for final filling:
_______________________________________
Date of filling:
_______________________________________
Number of vials after inspection:
Number of vials filled:
_______________________________________
Control tests on final lot A.6
Inspection of final containers
Appearance:
_______________________________________
Date of test:
_______________________________________
Results:
_______________________________________
Extractable volume
Extractable volume (ml): _______________________________________
The number of drops, using the approved
dropper, in a minimum of five individual
final containers:
_______________________________________
pH
Date of test:
_______________________________________
Result:
_______________________________________
Identity test
Page 80
Method used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Results:
_______________________________________
Lot number of reference reagents
_______________________________________
Tests for bacteria and fungi
Method used:
_______________________________________
Number of vials tested:
_______________________________________
Volume of inoculum per vial:
_______________________________________
Volume of medium per vial:
_______________________________________
Observation period (specification):
_______________________________________
Incubatio
n
Media used Inoculum Date of start of test Date of end of test Results
20–25 °C
__________ __________ ___________ ___________ __________
30–36 °C
__________ __________ ___________ ___________ __________
Negative
control __________ __________ ___________ ___________ __________
Virus titration
Date of test:
_______________________________________
Reference batch number:
_______________________________________
Titre of individual virus types:
_______________________________________
Batch numbers of antiserum used in test:
_______________________________________
Date of test:
_______________________________________
Results
Vaccine Reference
Type 1:
______________________ _______________________
Type 2:
______________________ _______________________
Type 3:
______________________ _______________________
Thermal stability
Date of test:
_______________________________________
Batch numbers of antiserum used in test
_______________________________________
Results: Vaccine at 37 ºC Vaccine Difference
Total virus:
______________ ______________ ______________
Page 81
Residual antibiotics (if applicable)
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Results
_______________________________________
Level of stabilizer (if applicable)
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Results:
_______________________________________
Additional information for production in monkey kidney-cell cultures
Production in monkey kidney-cell cultures
Control of vaccine production
_______________________________________
Control of monkeys
_______________________________________
Monkey species used for production:
_______________________________________
Quarantine batch number:
_______________________________________
Percentage of monkeys surviving quarantine
period: _______________________________________
Nature and concentration of antibiotics used in
the production cell culture maintenance medium: _______________________________________
Tests for antibodies to simian
immunodeficiency virus, SV40, foamy viruses
and B virus
Methods used:
_______________________________________
Date of start of test:
_______________________________________
Date of end of test:
_______________________________________
Results:
_______________________________________
Production details
Production monkey number:
_______________________________________
Date of trypsinizing:
_______________________________________
Number of cultures prepared:
_______________________________________
Cell cultures for vaccine production
Virus seed lot number:
_______________________________________
Virus titre/cell ratio: _______________________________________
Page 82
Number of cultures inoculated:
_______________________________________
Date of inoculation:
_______________________________________
Date of harvest:
_______________________________________
Temperature of incubation:
_______________________________________
Period of incubation:
_______________________________________
Number of cultures harvested:
_______________________________________
Tests on pooled supernatant fluids
Date of sampling from production
cell cultures: _______________________________________
Tests for adventitious agents:
_______________________________________
Volume tested/cell culture type:
_______________________________________
Observation period:
_______________________________________
Date of completion of tests:
_______________________________________
Results:
_______________________________________
Date of sampling from cell cultures inoculated
with the pooled fluid
_______________________________________
Tests for adventitious agents:
_______________________________________
Volume tested/cell culture type:
_______________________________________
Date of completion of tests:
_______________________________________
Results:
_______________________________________
Tests in rabbit kidney-cell cultures
Volume tested:
_______________________________________
Date of completion of tests:
_______________________________________
Results:
_______________________________________
Control of cell cultures
Ratio of control to production cell cultures or
control cell cultures as a proportion of production
cell cultures:
_______________________________________
Period of observation of cultures:
_______________________________________
Ratio or proportion of cultures discarded for
nonspecific reasons: _______________________________________
Results:
_______________________________________
Page 83
Tests for haemadsorbing viruses
Methods:
_______________________________________
Results:
_______________________________________
Tests for other adventitious agents
_______________________________________
Methods:
_______________________________________
Results:
_______________________________________
Control of single harvests
Volume harvested:
_______________________________________
Date of sampling:
_______________________________________
Tests for bacteria, fungi, and mycoplasmas:
_______________________________________
Results:
_______________________________________
Tests on neutralized single harvests in monkey
kidney-cell and human cell cultures
Batch number of antiserum used:
_______________________________________
Volume tested:
_______________________________________
Date of starting primary cell culture tests:
_______________________________________
Period of observation:
_______________________________________
Date of sampling cell culture fluids:
_______________________________________
Period of observation:
_______________________________________
Date of completion of tests:
_______________________________________
Results:
_______________________________________
Control of monovalent bulk
Tests in rabbits
Number and weight of animals:
_______________________________________
Date of inoculation:
_______________________________________
Results of injection:
_______________________________________
Quantity injected:
_______________________________________
Results (survival numbers, etc.):
_______________________________________
Date of filtration of bulk:
_______________________________________
Page 84
Porosity of filters used:
_______________________________________
Date of sampling:
_______________________________________
Tests for retroviruses
Methods:
_______________________________________
Date:
_______________________________________
Results :
_______________________________________
Submission addressed to National Regulatory Authority
Name of head of production (typed) _____________________________
Certification by the person from the control laboratory of the manufacturing company taking
overall responsibility for the production and control of the vaccine:
I certify that lot no. ______________ of poliomyelitis vaccine (oral), whose number appears on
the label of the final container, meets all national requirements and/or satisfies Part A of the
Recommendations to assure the quality, safety and efficacy of live attenuated poliomyelitis
vaccine (oral).
Signature: __________________________________________________
Name (typed): ______________________________________________
Date: ____
Page 85
Appendix 6
Model certificate for the release of live attenuated poliomyelitis vaccine (oral)
by national regulatory authorities
Lot release certificate
Certificate no. ________________
The following lot(s) of live attenuated poliomyelitis vaccine (oral) produced by
____________________________(1)
in _______________(2)
whose numbers appear on the
labels of the final containers, comply with the relevant specification in the marketing
authorization(3)
and the provisions for the release of biological products and Part A(4)
of WHO’s
Recommendations to assure the quality, safety and efficacy of live attenuated poliomyelitis
vaccines (oral) (_____)(5)
, and comply with WHO good manufacturing practices: main
principles for pharmaceutical products(6)
, Good manufacturing practices for biological
products(7)
and Guidelines for independent lot release of vaccines by regulatory authorities(8)
.
The release decision is based on _____________________________________________(9)
.
The certificate may include the following information:
• Name and address of manufacturer
• Site(s) of manufacturing
• Trade name and common name of product
• Marketing authorization number
• Lot number(s) (including sub-lot numbers and packaging lot numbers if necessary)
• Type of container
• Number of doses per container
• Number of containers/lot size
• Date of start of period of validity (e.g. manufacturing date) and/or expiry date
• Storage condition
• Signature and function of the authorized person and authorized agent to issue the certificate
• Date of issue of certificate
• Certificate number.
The Director of the National Regulatory Authority (or authority as appropriate):
Name (typed) _______________________________________________
Signature __________________________________________________
Date ______________________________________________________
Footnotes:
1 Name of manufacturer.
2 Country of origin.
3 If any national requirements are not met, specify which one(s) and indicate why release of the lot(s) has
nevertheless been authorized by the NRA.
4 With the exception of provisions on distribution and shipping, which the NRA may not be in a position to assess.
5 WHO Technical Report Series, No. (xx, xxxx).
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6 WHO Technical Report Series, No. 961, 2011, Annex 3.
7 WHO Technical Report Series, No. 822, 1992, Annex 1.
8 WHO Guidelines for Independent Lot Release of Vaccines by Regulatory Authorities adopted by the Sixty-first
meeting of the WHO Expert Committee on Biological Standardization in 2010.
9 Evaluation of summary protocol, independent laboratory testing, and/or specific procedures laid down in defined
document etc., as appropriate.
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Appendix 7
Preparation of live attenuated poliomyelitis vaccine (oral) using monkey
kidney-cell cultures
Example of a flowsheet of tests in cell cultures
HAEM = test for haemadsorbing viruses.
MK = monkey kidney cells from species (but not the same animal) used for production.
VK = kidney cells from vervet monkey or one sensitive to SV40 virus.
RK = rabbit kidney cells.
HC = human cells sensitive to measles.
Note: This example includes all tests, whether obligatory or not. Since the requirements applicable in a particular place
are those authorized by the NRA, this flowsheet should not be considered as an integral part of the requirements and has
been included solely for guidance. Manufacturing establishments should prepare their own flowsheet in order to clarify
the procedures used.